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Marine  Biological  Laboratory  Library 

Woods  Hole,  Mass. 


Presented  by 

John  Wiley  and  Sons  Inc» 
New  York  City 






















General  Editors:  sir  rudolph  peters,  f.r.s. 
and  F.  G.  YOUNG,  f.r.s. 


The  Biochemistry 
of  Semen 


M.D.,  Sc.D.,  Ph.D.,  F.R.S. 

Reader  in  Physiology  of  Animal  Reproduction, 
in  the  University  of  Cambridge 



First  published  in  1954 


CATALOGUE   NO.   4140    U  (NfETHUEN) 
Printed  and  Bound  in  Great  Britain  by  Butler  &  Tanner  Ltd.,  Fiome  and  London 

^"^-^  y^ 

LIBS  #. 


When  I  took  up  my  studies  on  semen  in  1944,  on  behalf  of  the 
Agricultural  Research  Council,  I  became  painfully  aware  of  the 
fact  that  information  on  the  physiology  of  semen,  its  chemical 
aspects  in  particular,  is  rather  difficult  to  come  by;  the  older 
observations  and  records  being  hidden  away  in  books  and 
journals  not  readily  accessible  in  any  but  the  best  equipped 
libraries,  and  moreover,  scattered  throughout  an  exceptionally 
wide  range  of  publications,  which  embrace  disciplines  as  far 
apart  as  say,  agriculture,  urology  and  cytology.  Judging  from 
numerous  requests  for  information,  received  from  fellow 
workers  in  the  field,  biochemists,  clinicians,  zoologists  and 
veterinary  officers  alike,  the  absence  of  a  fairly  comprehensive 
and  up-to-date  treatise  on  the  chemical  physiology  of  semen 
must  have  proved  a  serious  handicap  to  many  in  their  scientific 
and  practical  pursuits.  Therefore,  I  accepted  gladly  the  invita- 
tion to  write  this  book;  having  agreed  to  produce  but  a  'little 
book',  I  have  often  found  it  rather  irksome  to  condense  the 
vast  mass  of  data  into  the  allotted  space;  had  it  not  been  for 
the  encouragement  and  ready  help  of  colleagues — my  wife  not 
least  among  them,  the  task  would  have  been  even  more 

Biochemistry  of  semen  is  a  relatively  modern,  but  rapidly 
expanding,  field  of  physiology;  consequently,  many  of  our 
present  views,  particularly  as  regards  the  biological  significance 
of  various  chemical  constituents  of  semen,  may  have  to  be 
revised  or  modified  in  the  near  future.  That  being  so,  I  like  to 
look  upon  this  book,  or  at  any  rate,  those  parts  of  it  which 
deal  with  the  newer,  still  fluid  concepts,  as  something  in  the 
nature  of  an  Interim  Report,  designed  to  furnish  information 
and  to  convey  ideas  emerging  from  the  state  of  knowledge  as 
available  at  the  time  of  writing,  however  imperfect  that  may  be. 
In  presenting  the  recently  acquired  evidence,  I  have  tried  to 
render  justice  to  developments  in  the  sphere  of  mammalian  as 

vi  The  Biochemistry  of  Semen 

well  as  non-mammalian  physiology,  selecting  examples  from 
species  as  far  apart  as  man  and  the  sea-urchins,  and  occasion- 
ally, introducing  plants  as  well.  I  have  done  my  best  to  dis- 
tinguish between  established  fact  and  tentative  hypothesis,  and, 
as  far  as  possible,  have  refrained  from  the  tendency,  currently 
prevalent  among  workers  in  this  field,  to  assign  to  every  newly 
discovered  chemical  constituent  of  semen  a  major  role  in  the 
process  of  fertilization. 

I  wish  to  acknowledge  gratefully  the  help  of  those  who  gave 
me  permission  to  reproduce  plates  and  figures.  In  particular  1 
wish  to  extend  my  thanks  to  Dr.  C.  R.  Austin  (Sydney),  Dr. 
J.  L.  Hancock  (Cambridge)  and  the  Cambridge  University 
Press  for  Plate  I,  to  Prof.  L.  H.  Bretschneider  and  Dr.  Woutera 
van  Iterson  (Utrecht)  and  the  Nederland  Academy  of  Science 
for  Plate  II,  to  the  Royal  Society  for  Plate  III,  to  Lord  Roth- 
schild (Cambridge)  for  Plate  IV  and  for  reading  the  manuscript, 
to  the  Royal  Society  of  Edinburgh  for  Fig.  2,  to  Dr.  E.  Blom 
(Copenhagen)  and  the  Skandinavisk  Veterinartidskrift  for  Fig.  3, 
to  Dr.  C.  Huggins  (Chicago)  and  the  Harvey  Society  of  New 
York  for  Fig.  5,  to  Dr.  L.  Jacobsson  (Goteborg)  and  the  Acta 
Physiologica  Scandinavica  for  Fig.  11,  and  to  the  Cambridge 
University  Press,  Messrs.  Churchill  and  Messrs.  Macmillan  for 
permission  to  reproduce  Figs.  6-10,  12-14  and  16,  from  the 
Biochemical  Journal,  the  Journal  of  Agricultural  Science,  and 
Nature,  and  Plate  IV,  from  theCiba  Foundation  Symposium  on 
Mammalian  Germ  Cells.  I  should  also  like  to  thank  Miss  P.  A. 
Northrop  for  helping  me  in  the  preparation  of  the  typescript. 







Spermatozoa.  Spermatogenesis  and  sperm  'ripening'.  Sperm 
transport  in  the  female  reproductive  tract  and  'capacitation'. 
Structural  and  chemical  characteristics  of  the  sperm-head, 
middle-piece  and  tail. 

Seminal  plasma.  Secretory  function  of  male  accessory 
glands.  Prostatic  secretion.  Seminal  vesicle  secretion.  Physio- 
logical significance  of  seminal  plasma.  Prostaglandin,  vesi- 
glandin,  and  certain  other  pharmacodynamically  active  sub- 
stances. Coagulation  and  liquefaction. 



Species  and  individual  variations  in  the  composition  of 
semen.  Pre-sperm,  sperm-containing,  and  post-sperm  fractions 
in  the  ejaculate.  Criteria  for  the  rating  of  semen  quality. 
Optical  and  electrical  properties  of  semen.  Viscosity,  specific 
gravity,  osmotic  pressure,  and  ionic  equilibrium.  Hydrogen 
ion  concentration  and  buffering  capacity.  Metabolism  of 
semen  and  its  relation  to  sperm  density  and  motility;  glycolysis; 
methylene-blue  reduction  test;  respiration. 



Sperm  inhibitors  and  spermicidal  substances.  Chemical 
aspects  of  short-wave  radiation.  Variations  in  hydrogen  ion 
concentration  and  tonicity.  Influence  of  heat  and  cold;  sperm 
vitrification  and  'la  vie  latente'.  Role  of  hormones.  Sperm-egg 
interacting  substances  and  chemotaxis.  'Dilution  effect'  and 
chemical  changes  associated  with  senescence.  The  use  of 
artificial  diluents  in  the  storage  of  semen. 



viii  The  Biochemistry  of  Semen 



Mechanical  separation  of  sperm  from  seminal  plasma; 
release  of  intracellular  proteins  from  damaged  spermatozoa. 
Removal  of  the  sperm  nucleus  from  the  cytoplasm.  Protein- 
bound  iron,  zinc,  and  copper.  Cytochrome.  Catalase.  Hyal- 
uronidase  and  other  'lytic'  agents.  Sperm  nucleoproteins. 
Deoxyribonucleic  acid.  The  basic  nuclear  proteins;  protamines 
and  histones.  The  non-basic  nuclear  proteins;  karyogen  and 
chromosomin.  Keratin-like  protein  of  the  sperm  membrane. 


PLASMA  1 1 1 

Proteoses  and  free  amino  acids.  Fibrinolysin  and  fibrino- 
genase.  Pepsinogen.  Ammonia  formation.  Amino  acid  oxidase. 
Seminal  phosphatases;  'acid'  and  'alkaline'  phosphatase; 
5-nucleotidase;  pyrophosphatase.  Enzymic  hydrolysis  of 
adenosine  triphosphate. 


Lipids  in  spermatozoa.  The  lipid  capsule.  Acetal  phospho- 
lipids or  plasmalogens.  Role  of  lipids  in  sperm  metabolism. 
Lipids  in  the  seminal  plasma  and  male  accessory  gland 
secretions.  'Lipid  bodies'  and  prostatic  calculi. 


Fructose  as  a  normal  constituent  of  semen.  Species  differ- 
ences. Site  of  formation.  Seminal  fructose  as  an  indicator  of 
male  sex  hormone  activity;  the  'fructose  test'  and  its  applica- 
tion to  certain  problems  of  sex  endocrinology.  Role  of  hypo- 
physis. The  relationship  between  blood  glucose  and  seminal 
fructose.  Effect  of  malnutrition.  The  enzymic  mechanism  of 
fructose  formation.  Anaerobic  and  aerobic  utilization  of 
carbohydrate  by  spermatozoa.  Pasteur  effect  and  the  'meta- 
bolic regulator'.  Intermediary  reactions  in  sperm  fructolysis 
and  the  role  of  phosphorus-containing  coenzymes. 



Spermine.  Occurrence  of  crystalline  spermine  in  human 
semen;  its  chemical  nature  and  properties.  Derivatives  of 
spermine  and  their  use  in  forensic  medicine.  Synthesis  of 

Contents  ix 


spermine.  Spermidine.  Oxidation  of  spermine  and  spermidine 
by  diamine  oxidase.  State  of  spermine  in  semen. 

Choline.  The  Florence  reaction  in  semen.  Enzymic  libera- 
tion of  choline  from  precursors  in  semen.  Phosphorylcholine 
and  glycerylphosphorylcholine.  Physiological  function  of  free 
and  bound  choUne.  Choline  esterase. 

Ergothioneine.  Isolation  of  ergothioneine  from  the  boar 
seminal  vesicle  secretion.  The  function  of  seminal  ergothio- 
neine and  its  behaviour  towards  sulphydryl-binding  sub- 
stances. Biogenesis  of  ergothioneine. 

Creatine  and  creatinine.  Occurrence  in  mammalian  semen 
and  in  the  sperm  and  gonads  of  invertebrates.  Phosphocreatine 
and  phosphoarginine. 

Adrenaline  and  noradrenaline.  Occurrence  in  semen  and 
accessory  organs.  Enzymic  oxidation.  Pharmacodynamic 


Citric  acid.  Occurrence  and  distribution.  Influence  of  male 

sex  hormone.  Citric  acid  in  the  female  prostate.  Metabolism 

and  role  of  seminal  citric  acid. 
Inositol.  Occurrence  and  distribution,  m^^olnositol  as  a 

major  constituent  of  the  seminal  vesicle  secretion  in  the  boar. 

Physiological  function.  Relation  to  other  seminal  constituents. 



INDEX  223 



between  pages  1 0  and  1 1 

facing  page  14 










1  Diagrammatic  representation  of  a  spermatozoon  4 

2  'Ripening'  process  in  the  epididymis  of  the  mouse  7 

3  Schematic  representation  of  the  head  of  a  bull  spermato- 

zoon before  and  after  detachment  of  the  galea  capitis  13 

4  Diagrammatic  outline  of  male  accessory  organs  to  illustrate 

the  localization  of  fructose  16 

5  Diagram  of  osmotically  active  substances  in  prostatic 

fluid  18 

6  Relation  between  volume  of  ejaculate  and  content  of 

fructose  and  citric  acid  in  rabbit  semen  31 

7  Composition  of  boar  semen  fractions  collected  by  the 

'split-ejaculate  method'  at  half-a-minute  intervals  37 

8  Fructolysis  in  bull  semen  incubated  at  37°  47 

9  Effect  of  fluoride  on  the  respiration  and  aerobic  fructolysis 

of  ram  semen  51 

10  Effect  of  fructose,  glucose  and  lactate  on  the  respiration 

of  washed  ram  spermatozoa  53 

11  Increase    of   non-protein   nitrogen   and   amino-nitrogen 

content  in  human  semen  on  incubation  at  37°  112 

12  Post-castrate  fall  and  testosterone-induced  rise  of  seminal 

fructose  in  rabbit  140 

13  Dosage-response  curves  of  testosterone  propionate,  using 

the  coagulating  glands  of  the  rat  141 

14  Effect  of  alloxan  diabetes  and  insulin  on  seminal  fructose 

in  rabbit  147 

15  Diagrammatic  representation  of  fructolysis  in  semen  156 

16  Effect  of  ergothioneine  on  boar  spermatozoa  177 



Before  I  decided  to  embark  upon  the  business  of  studying  the 
metabolism  of  semen,  my  interest  used  to  centre  on  very  different 
biochemical  problems;  earlier  on,  in  the  laboratory  of  J.  K.  Parnas, 
I  was  youthfully  grappling  with  the  intricacies  of  intermediary 
carbohydrate  metabolism  in  muscle,  blood  and  yeast;  later  on,  at 
the  Molteno  Institute,  in  happy  association  with  D.  Keilin,  we  were 
investigating  the  nature  and  function  of  metalloprotein  enzymes  in 
plant  and  animal  tissues.  When  confronted  with  the  opportunity  of 
an  extensive  study  of  spermatozoa,  I  did  not  hesitate  to  give  up  my 
former  pursuits  in  order  to  devote  myself  to  experiments  involving 
biological  material  which  offers  the  investigator  a  chance,  almost 
unique  so  far  as  mammalian  tissues  are  concerned,  of  correlating 
chemical  and  metabolic  findings  with  clearly  defined  and  highly 
specific  criteria  of  physiological  activity,  such  as  the  motility  and 
fertilizing  capacity  of  the  spermatozoa.  Among  other  peculiarities 
which  make  semen  such  a  fascinating  and  attractive  object  of  study 
is  that  it  represents  an  animal  tissue  with  but  a  single  type  of  cells, 
the  spermatozoa,  freely  suspended  in  a  fluid  medium  of  some  com- 
plexity, the  seminal  plasma,  and  not  subject  to  cellular  growth, 
division  or  multiplication;  thus,  making  it  feasible  to  express  all 
one's  metabolic  measurements  directly  in  terms  of  cell  numbers, 
without  recourse  to  cumbersome  and  often  unreliable  standards  such 
as  dry  weight  of  tissue,  nitrogen  content,  or  indeed,  any  other  of  the 
commonly  used  metabolic  indices.  From  the  purely  practical  point 
of  view,  which  matters  greatly,  the  ability  of  spermatozoa  to  'survive', 
i.e.  retain  their  remarkable  properties  under  conditions  of  long-term 
storage  in  vitro,  is  of  great  importance.  This  in  turn,  gives  one  a 
chance  of  exploring  at  will  and  under  well-defined  conditions  in 
vitro,  the  intricate  chemical  mechanism  underlying  the  viability,  and 
ultimately,  the  senescence,  of  living  animal  cells. 

So  far  as  the  nutrition  of  spermatozoa  is  concerned,  semen 
resembles  more  a  suspension  of  microorganisms  in  a  nutrient 
medium,  than  other  animal  tissues  which  rely  for  their  nutrients 


xiv  The  Biochemistry  of  Semen 

on  the  blood  supply.  Nature  has  endowed  the  spermatozoa  with  the 
means  of  very  efficient  utilization  of  extraneous  sources  of  energy, 
such  as  are  accessible  to  the  sperm  cells  either  in  their  natural 
environment,  the  seminal  plasma,  or  in  the  artificial  storage  media. 

As  will  be  evident  from  what  follows  later,  the  present  century 
has  witnessed  much  that  is  new  in  the  field  of  semen  biochemistry. 
By  and  large,  however,  the  situation  is  not  very  different  from  what 
it  was  two  centuries  ago,  when  Charles  Bonnet  addressed  the  follow- 
ing remarks  about  spermatozoa  to  Spallanzani: 

'They  are,  of  all  animalculi  of  liquids,  those  which  have  most 
excited  my  curiosity:  the  element  in  which  they  live,  the  place  of 
their  abode,  their  figure,  motion,  their  secret  properties;  all,  in  a 
word,  should  interest  us  in  so  singular  a  kind  of  minute  animated 
beings.  How  are  they  found  there,  how  are  they  propagated,  how 
are  they  developed,  how  are  they  fed,  and  what  is  their  motion? 
What  becomes  of  them  when  the  liquid  they  inhabit  is  reabsorbed 
by  the  vessels  and  returned  to  the  blood?  Why  do  they  appear  only 
at  the  age  of  puberty;  where  did  they  exist  before  this  period?  Do 
they  serve  no  purpose  but  to  people  the  fluid  where  they  are  so 
largely  scattered?  How  far  are  we  from  being  able  to  answer  any  of 
these  questions!  And  how  probable  it  is,  that  future  age  will  be  as 
ignorant  of  the  whole,  as  our  own!' 


77?^  Two  Components  of  Semen: 
Spermatozoa  and  Seminal  Plasma 

Spermatozoa.  Spermatogenesis  and  sperm  'ripening'.  Sperm  transport  in 
the  female  reproductive  tract  and  'capacitation'.  Structural  and  chemical 
characteristics  of  the  sperm-head,  middle-piece  and  tail. 

Seminal  plasma.  Secretory  function  of  male  accessory  glands.  Prostatic 
secretion.  Seminal  vesicle  secretion.  Physiological  significance  of  seminal 
plasma.  Prostaglandin,  vesiglandin,  and  certain  other  pharmacodynamic- 
ally  active  substances.  Coagulation  and  liquefaction. 

'Whole  semen'  as  ejaculated,  generally  appears  as  a  viscous,  creamy, 
slightly  yellowish  or  greyish  fluid,  and  consists  of  spermatozoa  or 
'sperm',  suspended  in  the  fluid  medium,  called  seminal  plasma;  its 
composition  depends  in  the  first  place,  on  the  proportion  of  sperm 
and  plasma,  and  is  further  determined  by  the  size,  storage  capacity, 
and  secretory  output  of  several  different  organs  which  comprise  the 
male  reproductive  tract.  The  volume  of  the  ejaculate  and  the  con- 
centration of  spermatozoa  or  the  'sperm  density'  in  ejaculated  semen, 
vary  widely  from  one  species  to  another,  as  seen  from  Table  1.  A 
single  ram  ejaculate  for  instance,  amounts  to  0-7-2  ml.  only,  but  is 
distinguished  by  a  very  high  sperm  density,  2-5  million  per  f.i\. 
semen;  when  subjected  to  high-speed  centrifugation,  ram  semen 
separates,  on  the  average,  into  about  two-thirds  of  seminal  plasma 
and  one-third  of  firmly  packed  sperm.  Boar  semen  ejaculates  on  the 
other  hand,  may  reach  a  volume  of  as  much  as  500  ml;  this  is  not 
due  to  spermatozoa,  but  to  the  seminal  plasma  generated  in  very 
capacious  accessory  organs  (Plate  III);  a  sperm  density  not  exceed- 
ing 100,000  cells //^l.  is  quite  usual  for  boars,  and  even  lower  sperm 
densities  would  still  be  regarded  as  normal.  In  man,  the  average 
volume  of  a  single  ejaculate  is  about  3  ml.,  but  the  sperm  density 
is  frequently  less  than  100,000  cells///!.,  so  that  only  a  small  portion 
of  the  ejaculate,  much  less  than  10%,  is  represented  by  the  sperm 
and  the  rest  is  seminal  plasma. 


The  Biochemistry  of  Semen 

Table  1 .  Species  differences  in  volume  and  sperm  density  of 
ejaculated  semen 

Volume  of  single  ejaculate 

Sperm  density  in  semen 





Most  common 
value          1 

Normal  variations 

Average  value 



50           ' 

200  000-    600  000 

400  000 


0  05 

5  000  000-8  000  000 

6  000  000 




25  000-    300  000 

100  000 




300  000-2  000  000 

1  000  000 


0-2-1 -5 


50  000-6  000  000 

3  500  000 




1000  000-9  000  000 

3  000  000 




30  000-    250  000 

70  000 




50  000-    150  000 

100  000 




100  000-2  000  000 

700  000 




2  000  000-5  000  000 

3  000  000 




30  000-    800  000 

120  000 




7  000  000 

The  two  components  of  semen,  sperm  and  seminal  plasma,  differ 
in  their  origin,  composition  and  function,  and  must  be  considered 
separately,  in  much  the  same  sense  as  for  instance,  blood  corpuscles 
and  blood  plasma. 

Early  investigators  of  semen  were,  not  unnaturally,  fascinated  by 
the  spermatozoa,  and,  with  the  aid  of  such  optical  instruments  as 
were  available  to  them,  concentrated  their  efforts  upon  the  elucida- 
tion of  the  structural  details  of  spermatozoa.  But  it  is  very  much  to 
their  credit  that  they  have  not  entirely  neglected  the  seminal  plasma. 
Thus,  the  letter  in  which  Antoni  van  Leeuwenhoek  reported  in  1 677 
to  the  Royal  Society  on  sperm  motion,  also  contains  the  earliest 
description  of  spermin  crystals  in  the  seminal  plasma.  Louis  Nicolas 
Vauquelin,  the  author  of  the  first  treatise  on  the  chemical  composi- 
tion of  semen  (Experiences  sur  le  sperme  humain,  1791),  fully  appre- 
ciated the  separate  existence  of  sperm  and  seminal  plasma;  the  same 
was  true  of  his  followers,  among  them  Friedrich  Miescher,  whose 
collected  writings,  published  in  1897,  contained  much  new  informa- 
tion concerning  not  only  spermatozoa  but  the  seminal  plasma  as 

The  work  of  Miescher  and  his  contemporaries,  however,  dealt 
largely  with  fish  spermatozoa,  and  even  during  the  early  decades  of 

77?^  Two  Components  of  Semen  3 

the  present  century,  research  on  semen  was,  on  the  whole,  confined 
to  fish  and  generally  to  animals  in  which  fertilization  takes  place 
externally,  and  which  provide  the  experimental  material  in  con- 
veniently large  quantities.  The  tardy  progress  of  research  on  the 
spermatozoa  and  seminal  plasma  of  birds  and  mammals  was  due 
in  the  main  to  the  difficulty  of  securing  enough  material  for  experi- 
mental purposes;  however,  more  rapid  advances  were  made  soon 
after  Elie  Ivanov  (1907)  and  several  other  pioneers  in  the  field  of 
artificial  insemination,  perfected  the  technique  of  semen  collection 
from  domestic  animals.  The  widening  practice  of  artificial  insemina- 
tion for  breeding  purposes  on  a  large  scale,  early  revealed  the  need 
for  improved  standards  of  sperm  evaluation  and  in  this  way  pro- 
vided a  powerful  stimulus  for  morphological  as  well  as  chemical 
investigations  on  semen.  At  the  same  time,  clinical  enquiries  into  the 
causative  and  diagnostic  aspects  of  human  infertility  also  pointed 
to  serious  gaps  and  deficiencies  in  the  knowledge  of  the  physiology 
of  human  semen. 

The  last  two  decades  have  witnessed  rapid  advances  in  the  applica- 
tion of  laboratory  methods  of  semen  analysis  to  the  study  of  the 
manifold  causes  underlying  male  sterility  and  subfertility,  and  there 
is  a  steadily  increasing  number  of  publications  on  this  subject,  which 
has  been  comprehensively  reviewed  on  several  occasions.  Some  of 
these  articles  and  monographs  refer  specifically  to  man  (Joel,  1942; 
Hammen,  1944;  Hotchkiss,  1945;  Hinglais  and  Hinglais,  1947; 
Farris,  1950;  Lane-Roberts,  Sharman,  Walker,  Wiesner  and  Barton, 
1948;  Bayle  and  Gouygou,  1953;  Longo,  1953;  Williams,  1953),  while 
others  deal  with  various  animals  (Gunn,  1936;  Burrows  and  Quinn, 
1939;  Anderson,  1945;  Bonadonna,  1945;  Perry,  1945;  Walton,  1945; 
Milovanov  and  Sokolovskaya,  1947;  Van  Drimmelen,  1951;  Millar 
and  Ras,  1952).  In  addition,  much  valuable  information  on  sperm 
physiology  in  general,  indispensable  alike  to  those  engaged  in  human 
and  in  animal  research,  will  be  found  in  the  writings  of  Marshall 
(1922),  Hartman  (1939),  Chang  and  Pincus  (1951)  and  Walton  (1954), 
as  well  as  in  the  published  records  of  various  symposia  and  con- 
ferences held  under  the  auspices  of  such  bodies  as  the  Biochemical 
Society  {Biochemistry  of  Fertilization  and  the  Gametes,  1951),  the 
New  York  Academy  of  Sciences  {Biology  of  the  Testes,  1952),  the 
Ciba  Foundation  {Mammalian  Germ  Cells,  1953),  the  National 

4  The  Biochemistry  of  Semen 

Committee  on  Maternal  Health  {Diagnosis  in  Sterility,  1946;  The 
Problem  of  Fertility,  1946;  Studies  on  Testis  and  Ovary,  Eggs  and 
Sperm,  1952),  the  American  Society  for  the  Study  of  Sterility 
(official  journal:  Fertility  and  Sterility)  and  the  British  Society  for  the 
Study  of  Fertility  {Proceedings). 


Spermatogenesis  and  sperm  'ripening'' 

Spermatozoa  (Plate  I  and  Fig.  1)  originate  in  the  testis  from  the 
germ  or  spermatogenic  cells  of  the  seminiferous  epithelium  in  the 

Galea  capitis 






Nucleus  (poscerior  part) 


Axial  filament 

Fig,  1.  Diagrammatic  representation  of  a  spermatozoon. 

course  of  spermatogenesis,  a  process  of  stepwise  proliferation  and 
transformation,  distinguished  by  the  successive  stages  of  sperma- 
togonia, primary  spermatocytes,  secondary  spermatocytes,  and 
spermatids.  The  present  knowledge  concerning  the  chemical  changes 

The  Two  Components  of  Semen  5 

which  take  place  during  spermatogenesis  is  defective  and  rests  almost 
entirely  on  histochemical  observations. 

In  several  species  so  far  investigated,  spermatogonia  and  sperma- 
tocytes have  been  shown  to  have  a  cytoplasm  which  is  basophilic, 
in  distinction  to  mature  spermatozoa  which  exhibit  only  a  faint 
coloration  of  the  flagellum.  Cytochemical  studies  carried  out  by 
Brachet  (1944,  1947)  have  shown  that  the  affinity  of  the  sperma- 
togonia and  spermatocytes  for  basic  dyes  is  due  to  ribonucleic  acid, 
and  cytochemical  as  well  as  spectrophotometric  studies  (Caspersson, 
1939)  point  to  the  fact  that  spermatogenesis  involves  a  progressive 
disappearance  of  ribonucleic  acid  from  the  developing  sperm  cell. 
In  ejaculated  spermatozoa  of  the  bull,  Vendrely  and  Vendrely  (1948) 
using  the  analytical  methods  of  Schmidt  and  Thannhauser  (1945) 
and  Schneider  (1945),  found  a  content  of  0-2x1 0~^  mg.  ribonucleic 
acid  per  sperm  cell,  that  is  fifteen  times  less  than  the  corresponding 
value  for  deoxyribonucleic  acid.  An  analysis  of  mature  ram  sperma- 
tozoa carried  out  in  our  laboratory  with  the  Markham-Smith 
chromatographic  procedure  (1949)  which  is  based  on  the  identifica- 
tion of  uridylic  acid  in  an  acid  hydrolysate  of  ribonucleic  acid, 
failed  to  reveal  the  presence  of  uridylic  acid.  As  to  the  origin  of 
ribonucleic  acid  in  the  spermatogonia  and  spermatocytes,  a  study  of 
the  spermatogenesis  in  Asellus  aquaticus  (Vitagliano  and  de  Nicola, 
1948)  suggests  that  ribonucleic  acid  is  not  elaborated  in  the  develop- 
ing gametes  themselves  but  is  secreted  by  the  surrounding  cells  and 
then  absorbed  and  utilized  by  the  germ  cells. 

Two  other  processes  associated  with  spermatocytic  development 
are:  the  progressive  decline  of  alkaline  and  acid  phosphatase  activity 
(assessed  histochemically)  in  the  nuclei  (Krugelis,  1942;  Wolf,  Kabat 
and  Newman,  1943),  and  a  simultaneous  disappearance  of  glycogen. 
Both  the  Sertoli  cells  and  the  spermatogonia  abound  in  glyco- 
gen, which  also  occurs,  although  in  a  smaller  concentration,  in  the 
primary  spermatocytes  (Montagna  and  Hamilton,  1951;  Elftman, 
1952;  Long  and  Engle,  1952;  Mancini,  Nolazco  and  Baize,  1952). 
But  the  secondary  spermatocytes  and  the  spermatids  give  practically 
no  cytochemical  reactions  for  glycogen,  and  in  the  mature  sperma- 
tozoa the  glycogen  content  is  exceedingly  low:  in  ejaculated  ram 
semen  'glycogen'  content,  i.e.  the  alkali-resistant  polysaccharide 
which  behaves  like  glycogen  on  ethanol-precipitation  and  which 

6  The  Biochemistry  of  Semen 

yields  on  hydrolysis  glucose  (as  determined  by  glucose  oxidase) 
seldom  exceeds  01%,  and  may  be  as  little  as  0019%  (Mann,  \9A6b). 
Similarly,  in  sea-urchin  sperm  {Echinus  esculentus),  the  ethanol- 
precipitable,  glycogen-like  material  separated  from  sperm  and 
analysed  after  acid  hydrolysis  by  means  of  glucose  oxidase,  repre- 
sents no  more  than  004%  on  a  wet-weight  basis  (Rothschild  and 
Mann,  1950).  Even  oysters,  in  which  as  much  as  one-third  of  the  dry 
body  weight  may  consist  of  glycogen,  produce  spermatozoa  which 
when  ripe,  contain  no  more  than  1  %  glycogen  on  a  dry-weight  basis 
(Humphrey,  1950). 

Yet  another  phenomenon  accompanying  spermatogenesis  is  a 
significant  change  in  the  distribution  of  lipids.  In  the  deer  (Wislocki, 
1949)  and  in  the  rat  (Lynch  and  Scott,  1951),  sudanophilic  material 
is  concentrated  chiefly  in  the  Sertoli  cells,  but  in  man  (Montagna, 
1952)  a  high  content  of  lipids  is  characteristic  alike  of  the  Sertoli 
cells  as  well  as  the  cytoplasm  of  spermatogonia  and  of  primary 
spermatocytes.  Similarly,  in  certain  invertebrates,  as  for  example, 
Lithobius  forficatiis  (Monne,  1942),  lipids  form  a  highly  characteristic 
component  of  the  cytoplasm  in  spermatocytes.  These  cytoplasmic 
lipids  are  usually  birefringent  and  give  positive  reactions  for  steroids. 
In  ejaculated  spermatozoa,  at  any  rate  those  of  mammals,  the  lipids 
are  confined  largely  to  certain  definite  regions  such  as  the  'mito- 
chondrial sheath'  of  the  middle-piece  and  the  so-called  lipid  capsule; 
these  will  be  described  in  more  detail  later. 

The  changes  initiated  by  spermatogenesis  continue  during  the  stay 
of  spermatozoa  in  the  epididymis,  and  form  a  part  of  the  'ripening' 
process.  The  metabolism  of  epididymal  spermatozoa  which  are  often 
immotile,  but  capable  of  long  survival,  is  as  yet  only  poorly  under- 
stood. Guinea-pigs  and  rabbits,  for  example,  can  remain  fertile 
for  some  weeks  after  the  ligation  of  the  ductuli  efferentes,  and  in  bats 
spermatozoa  have  been  detected  in  the  cauda  epididymis  as  late  as 
seven  months  after  the  cessation  of  spermatogenesis. 

A  striking  change  associated  with  the  process  of  sperm  ripening 
in  the  epididymis  is  the  migration  of  a  drop-like  swelling  of  sperm 
cytoplasm  called  the  'kinoplasmic  droplet'  and  believed  to  contain 
some  lipid  material;  when  one  examines  spermatozoa  from  the  caput 
epididymidis  of  a  mouse  for  example,  the  kinoplasmic  droplet  is 
usually  situated  close  to  the  proximal  (anterior)  end  of  the  middle- 

The  Two  Components  of  Semen  1 

piece,  but  by  the  time  the  spermatozoa  have  reached  the  cauda 
epididymidis  and  are  nearing  the  vas  deferens,  the  droplets  take  up 
a  position  at  the  distal  (posterior)  end  of  the  middle-piece  (Merton, 
1939;  Fig.  2).  Finally,  they  tend  to  disappear  altogether  and  are 
seldom  found  in  ejaculated  sperm,  except  m  certain  abnormal  cases 
(Plate  I).  Some  authors  regard  the  kinoplasmic  droplet  as  no  more 
than  a  remnant  of  spermatid  cytoplasm  devoid  of  special  signi- 
ficance, but  there  are  those  who  believe  that  it  plays  an  important 
role  by  nourishing  the  spermatozoon  during  the  passage  through  the 
epididymis,  before  the  sperm  cells  establish  contact  with  an  extra- 
cellular source  of  nutrient  material,  in  the  form  of  seminal  plasma. 
The  disappearance  of  the  kinoplasmic  droplet  is  but  the  final 
stage  in  the  process  of  gradual  shrinkage  and  'dehydration'  of  proto- 
plasm which  accompanies  both  spermatogenesis  and  ripening,  and 
from  which  the  'ripe'  spermatozoon  ultimately  emerges  as  a  cell 
with  a  highly  condensed  nucleus  and  very  little  cytoplasm.  Associated 
with  the  diminution  of  protoplasm  is  a  progressive  loss  of  water 
and  a  corresponding  increase  in  the  specific  gravity  of  the  sperm  cell. 
Lindahl  and  Kihlstrom  (1952)  suspended  equal  numbers  of  bull 
spermatozoa  in  a  series  of  aqueous  solutions  of 
the  methylglucamine  salt  of  'umbradil'  (2  :  5-di- 
iodine-4-pyridone-A^-acetic  acid),  the  lightest  of 
which  (sp.  g.  10918,  osmotic  pressure  18  atm.) 
had  a  lower  specific  gravity  than  any  of  the 
spermatozoa,  the  heaviest  (sp.  g.  1-3519,  osmotic 
pressure  220  atm.)  being  of  about  the  same 
specific  gravity  as  the  'densest'  spermatozoa; 
these  sperm  suspensions  were  centrifuged  in 
haematocrit  tubes  so  that  all  spermatozoa  with 
a  specific  gravity  exceeding  that  of  the  medium, 
formed  a  sediment  in  the  graded  capillary  part  of 
haematocrit  tubes.  The  specific  gravity  of  bull 

Fig.  2.  ''Ripening''  process  in  the  epididymis  of  the 
mouse;  (a)  spermatozoon  from  the  caput  epi- 
didymis with  proximal  kinoplasmic  droplet; 
(b)  spermatozoon  from  the  cauda  epididymis 
with  distal  kinoplasmic  droplet. 

(Merton,  1939) 


8  The  Biochemistry  of  Semen 

spermatozoa  determined  in  this  manner  ranged  from  1-240  to  1-334; 
there  was  a  negative  correlation  between  the  mean  specific  gravity 
and  the  percentage  of  unripe  spermatozoa,  that  is  those  which 
still  possessed  the  kinoplasmic  droplet;  in  each  experiment,  the 
concentration  of  unripe  spermatozoa  was  significantly  higher  in 
the  'floating',  than  in  the  sedimenting,  fraction,  the  specific  gravity 
of  the  unripe  sperm  cells  being  less  than  that  of  the  ripe  ones. 

To  some  extent,  the  specific  gravity  of  spermatozoa  may  be 
accounted  for  by  the  high  concentration  of  deoxyribonucleoprotein 
in  the  sperm  nucleus,  but  in  a  large  measure  it  is  also  due  to  the  state 
of  'dehydration'  which  is  characteristic  of  the  sperm  protoplasm 
and  its  protein  constituents.  Hand  in  hand  with  the  high  specific 
gravity  goes  a  remarkably  high  refractive  index  and  light-reflection 
power  of  the  spermatozoa.  In  general,  the  refractive  index  of  most 
living  animal  cells  lies  between  1-350  and  1-367,  corresponding  to  a 
10-20%  concentration  of  solids;  but  in  human  spermatozoa  exam- 
ined by  the  immersion  method,  Barer,  Ross  and  Tkaczyk  (1953) 
obtained  values  corresponding  to  a  content  of  almost  50%  solids. 
Nephelometric  measurements  of  light  reflection  carried  out  with 
bull  semen  samples  containing  a  varying  percentage  of  'unripe' 
spermatozoa,  showed  that  the  capacity  of  the  sperm  cell  to  reflect 
light  increases  with  ripening  (Lindahl,  Kihlstrom  and  Strom,  1952); 
there  appears  to  be  a  close  relationship  between  the  light-reflecting 
power  of  sperm  and  the  characteristic  'luminosity'  of  the  surface 
of  spermatozoa  under  dark-field  illumination,  which,  in  all  prob- 
ability, is  due  to  the  'waterlessness'  of  the  lipid  capsule  surrounding 
the  ripe  sperm  cell. 

Sperm  transport  in  the  female  reproductive  tract  and  ' capacitation' 

There  is  evidence  that  the  process  of  sperm  ripening  is  not  halted 
at  ejaculation  but  proceeds  in  the  female  reproductive  tract,  where 
the  sperm  cell  undergoes  a  definite  change,  called  capacitation, 
before  it  becomes  capable  of  penetrating  the  egg  surface  (Austin, 
1951;  Chang,  1951;  Austin  and  Braden,  1952;  Thibault,  1952).  It  is 
quite  likely  that  the  success  which  some  early  investigators  had  in 
achieving  fertilization  with  artificially  inseminated  epididymal  sper- 
matozoa, was  due  to  the  continuation  of  sperm  ripening  processes 
in  the  female  reproductive  tract. 

The  Two  Components  of  Semen  9 

Whether  the  semen  is  ejaculated  into  the  uterus  (sow),  or  into  the 
cervix  or  vagina  (cow,  rabbit),  a  certain  time  is  always  required  for 
the  passage  of  spermatozoa  to  the  oviducts  and  for  their  accumula- 
tion in  adequate  numbers  at  the  site  of  fertilization.  The  time  needed 
for  some  of  the  spermatozoa  at  any  rate,  to  arrive  at  their  goal  may 
be  relatively  short;  a  quarter  of  an  hour  or  less,  in  the  rat  (Blandau 
and  Money,  1944),  cow  (VanDemark  and  Moeller,  1951)  and  ewe 
(Starke,  1949;  Dauzier  and  Wintenberger,  1952);  a  matter  of  a  few 
minutes  in  the  rabbit  (Lutwak-Mann,  unpublished).  This  indicates 
that  in  these  animals  the  spermatozoa  are  conveyed  to  their  final 
destination  thanks  to  certain  concomitant  movements  of  the 
female  tract  and  do  not  depend  exclusively  upon  their  own  motility. 
However,  from  the  moment  of  arrival  in  the  ovarian  tube,  time 
must  elapse  before  the  sperm  cell  is  capable  of  fertilizing  the  egg. 
In  the  rabbit,  ovulation  takes  place  about  ten  hours  after  copulation, 
and  presumably,  spermatozoa  require  this  period  of  time  to  undergo 
complete  'capacitation'.  As  Chang  (1951)  has  shown,  rabbit  sperma- 
tozoa placed  in  the  Fallopian  tubes  soon  after  ovulation,  penetrate 
a  larger  proportion  of  eggs  if  they  had  been  previously  kept  for  about 
five  hours  in  the  uterus  of  another  doe.  According  to  Austin  (1951), 
rat  spermatozoa  injected  into  the  periovarian  sac  of  the  rat  after 
ovulation,  do  not  begin  to  enter  the  eggs  until  some  five  hours  later. 
The  processes  of  sperm  maturation  and  capacitation  are  linked  in 
some  as  yet  not  fully  understood  manner,  with  the  survival  of  sperm 
in  the  female  tract.  In  higher  mammals  this  period  is  usually  limited 
to  one  or  two  days,  but  the  'longevity'  of  bird  sperm  is  remarkable, 
and  in  bats  and  the  terrestrial  isopode  Armadillidium  vulgare  the 
spermatozoa  are  said  to  survive  in  the  female  tract  for  many  months, 
in  certain  insects  even  for  years.  In  insects,  however,  this  striking 
behaviour  of  spermatozoa  is  probably  bound  up  closely  with  certain 
other  peculiarities  of  sperm  transport:  in  many  instances,  the  sperma- 
tozoa are  conveyed  to  the  female  not  in  a  free  fluid  medium,  but  are 
enclosed  in  a  sac  or  'spermatophore'  which  is  deposited  in  the  'bursa 
copulatrix'  or  in  the  vagina;  from  there,  after  the  sac  has  been 
emptied,  they  move  on  to  the  'spermatheca',  a  pouch  which  serves 
as  a  special  storage  organ  for  the  spermatozoa,  where  they  remain 
till  the  time  of  fertilization. 

It  must  also  be  remembered  that  not  all  spermatozoa  present  in 

10  The  Biochemistry  of  Semen 

a  given  ejaculate  survive  for  the  same  length  of  time.  In  higher 
mammals  for  instance,  of  the  many  hundreds  of  millions  of  sperm 
cells,  only  a  minute  fraction,  not  more  than  a  few  thousand,  reach 
the  site  of  fertilization,  and  ultimately  only  a  single  spermatozoon  is 
responsible  for  the  fertilization  of  the  ovum. 

Structural  and  chemical  characteristics  of  the  sperm-head,  middle- 
piece,  and  tail 

In  the  majority  of  species,  including  man,  mature  spermatozoa 
have  a  filiform  structure  owing  to  the  presence  of  a  flagellate  append- 
age, although  non-flagellar  forms  of  sperm  cells  are  not  uncommon 
in  certain  lower  animals,  for  example,  among  Crustacea  and  nema- 
todes. This  peculiar  filiform  structure  determines  to  a  considerable 
extent,  the  remarkable  permeability  of  the  sperm  cell,  which  is  per- 
haps best  illustrated  by  the  so-called  'leakage'  phenomenon,  that  is, 
the  remarkable  ease  with  which  even  large  molecules  such  as  cyto- 
chrome c  or  hyaluronidase  can  detach  themselves  from  the  sperm 
structure  and  pass  into  the  extracellular  environment.  The  high 
degree  of  permeability  explains  the  speed  with  which  exchange  re- 
actions can  take  place  between  the  spermatozoa  and  the  surrounding 
medium,  whether  this  be  the  seminal  plasma  or  an  artificial  pabulum; 
moreover  it  makes  it  possible  for  certain  intermediary  enzymic 
reactions  such  as  those  involved  in  the  phosphorylative  breakdown 
of  carbohydrate,  to  be  demonstrated  directly  in  intact  spermatozoa, 
without  cell  disintegration  which  is  an  unavoidable  prerequisite  in 
studies  on  the  intermediary  enzymes  of  other  animal  tissues.  This 
does  not  necessarily  apply  to  all  enzymes  and  the  failure  to  demon- 
strate an  enzyme  in  intact  sperm  cells  must  not  be  taken  as  evidence 
of  its  absence,  particularly  so  in  the  case  of  mammalian  sperma- 
tozoa which  are  resistant  to  the  action  of  most  plasmolysing  agents, 
including  water. 

The  principal  morphological  features  of  spermatozoa  have  been 
established  largely  in  the  last  century  with  the  help  of  the  ordinary 
light  microscope,  by  pioneers  such  as  Ballowitz,  Jensen,  Meves, 
Retzius  and  others,  but  many  more  details  have  emerged  since  as  a 
result  of  the  application  of  new  techniques,  particularly  those  of 
histochemistry  (Marza,  1930;  Popa  and  Marza,  1931;  Brachet,  1944; 
Leblond,    Clermont    and    Cimon,     1950;     Leuchtenberger    and 




a.  Normal  bull  semen;  photographed  in  ultraviolet  light  at  2750  A. 

Mag.  X  2700. 
h.  Semen  from  an  infertile  bull;  the  spermatozoa  are  'unripe'  and  show 

kinoplasmic  droplets  at  the  anterior  ends  of  the  middle-pieces; 

nigrosin-eosin  stain. 


c.  Rat  spermatozoon. 

d.  Rat  spermatozoon  with  a  kinoplasmic  droplet  at  the  posterior  end  of 

the  middle-piece. 

e.  Rat  spermatozoon.  Mag.  x  1500. 

(By  courtesy  of  Dr.  C.  R.  Austin  and  Dr.  J.  L.  Hancock) 

The  Two  Components  of  Semen  11 

Schrader,  1950;  Wislocki,  1950;  Friedlaender  and  Fraser,  1952; 
Hancock,  1952;  Melampy,  Cavazos  and  Porter,  1952)  and  electron 
microscopy  (Seymour  and  Benmosche,  1941;  Harvey  and  Anderson, 
1943;  Schmitt,  1944;  Bretschneider  and  Iterson,  1947;  Bretschneider, 
1949^,  b\  Grigg  and  Hodge,  1949;  Hodge,  1949;  Randall  and  Fried- 
laender, 1950;  Bayle  and  Bessis,  1951;  Friedlaender,  1952;  Challice, 
1953;  Bradfield,  1954). 

In  a  typical  flagellar  spermatozoon  (Plate  I  and  Fig.  1)  it  is  usually 
possible  to  distinguish  three  regions,  viz.  sperm-head,  middle-piece 
and  tail,  but  even  among  closely  related  species,  one  encounters  an 
extraordinary  diversity  of  form,  size  and  structure.  Moreover,  on 
examining  the  semen  from  a  single  individual,  one  often  finds  in 
addition  to  the  normally  shaped  spermatozoa,  a  variety  of  'degener- 
ate', 'abnormal'  or  'immature'  forms  which  represent  every  con- 
ceivable deviation  from  the  normal  cell  structure,  from  'tapering' 
and  'double'  cells  with  a  double  head  or  tail,  to  'giant'  and  'monster' 
cells  containing  several  nuclei  and  several  tails  in  a  mass  of  cyto- 
plasm. Although  a  high  degree  of  sperm  abnormality  is  undoubtedly 
associated  with  subfertility,  normal  semen  is  seldom  completely 
uniform,  and  human  semen  for  example,  is  reckoned  to  contain 
as  a  rule,  at  least  20%  of  abnormal  forms  (Pollak  and  Joel,  1939; 
Harvey  and  Jackson,  1945;  Hotchkiss,  1945;  Lane-Roberts  et  al., 
1948;  Williams,  1950).  In  the  bull  (Williams  and  Savage,  1927; 
Lagerlof,  1934;  Bishop,  Campbell,  Hancock  and  Walton,  1954) 
and  stallion  (Bielanski,  1951),  the  percentage  of  abnormal  forms  in 
semen  is  similarly  high,  in  the  ram  on  the  other  hand,  it  appears  to 
be  much  less. 

The  shape  of  the  head  in  a  normal  spermatozoon  varies  greatly; 
it  is  ovoid  in  the  bull,  ram,  boar,  and  rabbit,  it  resembles  an  elong- 
ated cylinder  in  fowl  and  has  the  form  of  a  hook  in  the  mouse  and 
rat;  in  the  human  species,  the  sperm-head  appears  as  a  flattened,  oval 
body,  about  4-6  ^  long,  2-6  /<  wide,  and  1-5  f-i  thick,  which  is  com- 
pressed at  the  anterior  pole  into  a  thin  edge. 

The  main  part  of  the  head  is  occupied  by  the  nucleus,  filled  by 
closely-packed  chromatin  which  consists  largely  of  deoxyribonucleo- 
protein  and  gives  a  positive  Feulgen  (nucleal)  reaction  with  Schiff"'s 
fuchsin-sulphurous  acid  reagent.  The  anterior  part  of  the  nucleus 
is  covered  by  a  cap-like  structure  known  as  the  acrosome.  The 

12  The  Biochemistry  of  Semen 

latter  gives  no  positive  Feulgen  reaction  but  stains  with  the  Schiff 
reagent  after  exposure  to  the  oxidizing  action  of  periodic  acid  as 
demonstrated  in  the  sperm  of  the  hemipteran  insect,  Arvelius  albo- 
punctatus  (Leuchtenberger  and  Schrader,  1950)  and  in  bull  sperm 
(Hancock,  1952).  According  to  McManus  (1946)  and  Hotchkiss 
(1948),  the  'periodic  acid  Schiff  reaction'  (PAS)  is  due  to  the  pre- 
sence of  carbohydrates,  and  the  chemical  groups  which  react  with 
fuchsin-sulphurous  acid  are  the  aldehydes  formed  from  1  :  2  glycol 
groupings  by  oxidation  with  periodic  acid: 
OH     OH 

I  I 

R— C C— R  +  HIO4  ->  2R— CHO 

The  acrosomal  material  is  not  glycogen  as  it  does  not  react  with 
iodine  and  is  not  affected  by  treatment  with  amylase.  It  cannot  be 
hyaluronic  acid  because  it  resists  the  action  of  hyaluronidase.  The 
possibility  that  it  may  be  related  to  hyaluronidase  itself  still  remains 
to  be  investigated.  There  has  also  been  a  tendency  to  regard  it  as  a 
mucopolysaccharide,  without  however,  sufficient  evidence.  Special 
precautions  are  called  for  in  the  preparation  of  spermatozoa  for  the 
PAS  reaction.  Structural  changes  in  sperm  cells,  such  as  occur  for 
example,  after  rapid  cooling  ('temperature  shock'),  may  render  the 
acrosomal  material  unresponsive  to  the  periodic  acid-Schiff  reagent. 
It  is  not  improbable  that  the  acrosomal  'polysaccharide'  is  either 
decomposed  or  detached  from  the  head  of  a  mature  spermatozoon; 
this  is  borne  out  by  some  microscopic  observations  on  changes  which 
take  place  in  the  acrosome  during  the  period  of  senescence  and  death 
of  the  sperm  cell.  Several  investigators  have  described  in  sperma- 
tozoa yet  another  cap,  a  loose  protoplasmic  structure,  named  'galea 
capitis'  (also  'acrosome  cap',  'Kopfkappe'  or  'capuchon  cephalique') 
which  envelops  the  apical  part  of  the  sperm-head  and  can  break 
away  spontaneously  to  form  a  so-called  'spermatic  veil'  or  'floating 
cap'  (Williams  and  Savage,  1925;  Blom,  1945).  However,  whereas 
most  authors  including  Williams  (1950)  regard  the  acrosome  proper 
and  the  galea  capitis  as  two  distinct  structural  entities,  some  con- 
sider them  to  be  identical,  and  Hancock  (1952)  for  instance,  is  con- 
vinced that  there  is  only  one  acrosomal  structure  and,  that  the  de- 
tachable cap  arises  through  post-mortem  changes,  and  is  the  result 
of  swelling  and  loosening  of  the  acrosome  itself.  The  separation  of 

The  Two  Components  of  Semen  1 3 

the  galea  capitis  can  be  conveniently  followed  by  the  India-ink 
staining  technique  of  Blom  (Fig.  3)  whose  studies  indicate  that  the 
phenomenon  occurs  most  frequently  in  degenerating  spermatozoa, 
for  example,  in  ejaculates  obtained  after  long  periods  of  abstinence. 
When  stained  by  the  Gomori  technique,  the  galea  capitis  or  at  least 
the  region  near  the  tip  of  the  head,  shows  a  positive  reaction  for  acid 
phosphatase.  On  the  whole,  however,  phosphatase  activity,  'acid' 
and  'alkaline'  alike,  is  much  more  intense  in  the  seminal  plasma 
than  in  the  spermatozoa.  Moreover,  as  it  is  rather  difficult  to 

Fig.  3.  Schematic  representation  of  the  head  of  a  bull  spermatozoon  before 
and  after  detachment  of  the  galea  capitis  {India-ink  method). 

a,  pars  posterior,  b,  pars  intermedia,  c,  pars  anterior  with  the  galea  capitis 

in  situ,  q,  the  'bare'  pars  anterior  after  detachment  of  the  galea,  Cg,  galea 

capitis  detached  from  the  head. 

(Blom,  1945) 

remove  from  the  spermatozoa,  even  by  exhaustive  washing,  all 
adhering  traces  of  seminal  plasma,  the  possibility  of  contamination 
with  plasma  phosphatase  must  be  taken  into  account  when  con- 
sidering the  occurrence  of  phosphatase  in  the  spermatozoa  themselves. 
The  narrow  region  which  connects  the  sperm-head  with  the 
middle-piece  is  known  as  the  neck  (or  neck-piece),  which  is  the  most 
vulnerable  and  fragile  part  of  the  spermatozoon.  The  entire  neck 
region  is  bounded,  however,  by  a  membrane  which  continues  over 
the  head  and  middle-piece.  In  the  neck,  close  to  the  base  of  the 
sperm  nucleus,  is  situated  the  centrosome  which  marks  the  beginning 
of  the  'axial  filament',  the  central  core  of  both  the  middle-piece  and 
tail.  The  axial  filament  consists  of  a  number  of  fine  long  fibrils  which 

14  The  Biochemistry  of  Semen 

run  uninterruptedly  through  the  whole  length  of  the  middle-piece 
and  tail.  These  fibrils  probably  represent  the  main  contractile  ele- 
ment of  the  sperm  cell,  responsible  for  the  whip-like  lashing  of  the 
tail.  In  most  species  investigated  so  far  by  means  of  the  electron 
microscope,  eleven  fibrils  have  been  identified;  two  of  these,  which 
occupy  the  central  position,  are  sensitive  to  the  action  of  water  and 
digestive  proteolytic  enzymes,  whereas  the  remaining  nine  fibrils 
which  form  an  'outer  cylinder'  around  the  'central  pair',  are  remark- 
ably resistant  to  the  action  of  plasmolysing  and  digestive  agents, 
and  even  prolonged  proteolysis  with  pepsin  or  trypsin  fails  to  disrupt 
them;  these  fibrils  also  resist  effectively  attempts  at  solubilization  by 
means  of  salt  solutions,  acids  and  weak  bases. 

The  finer  structure  of  the  individual  fibrils  is  still  a  matter  of  active 
investigation.  In  the  case  of  mammalian  spermatozoa,  doubling  of 
fibrils  has  been  observed,  at  any  rate  in  the  middle-piece,  and  in 
addition  to  the  outer  cylinder  of  nine  fibrils,  another,  so-called 
inner  cylinder  has  been  described,  consisting  of  nine,  much  thinner 
fibrils.  The  precise  chemical  nature  of  the  fibrillar  protein  is  un- 
known. A  certain  resemblance  to  muscular  contraction  prompted 
Engelhardt  (1946)  to  ascribe  to  the  contractile  substance  of  sperma- 
tozoa myosin-like  properties,  and  to  sperm  adenosine-triphos- 
phatase the  role  of  'spermosin'.  However,  this  c^aim  remains  at 
present  unsubstantiated  since  it  was  not  accompanied  by  satisfac- 
tory evidence  that  the  spermatozoa  used  for  the  experiments,  were 
really  free  from  phosphatases,  especially  the  powerful  adenosine- 
triphosphatase,  of  seminal  plasma. 

In  the  middle-piece  (or  midpiece)  which  in  the  human  sperma- 
tozoon is  about  the  length  of  the  sperm-head  though  only  one-tenth 
as  wide,  the  axial  filament  is  surrounded  by  the  'broad  helix',  also 
called  'spiral  body'  or  'mitochondrial  sheath'.  This  lipid-rich  struc- 
ture, which  is  believed  to  be  derived  from  mitochondria,  has  the 
shape  of  a  broad  paired  thread,  wound  helicoidally  round  the 
'outer  cylinder'  of  sperm  fibrils.  It  is  here  that  the  cytochrome- 
cytochrome  oxidase  system  of  spermatozoa  is  believed  to  be  con- 
centrated. The  junction  between  the  middle-piece  and  tail  is  marked 
by  the  presence  of  a  ring  centriole. 

The  tail  or  'flagellum'  in  the  human  spermatozoon  is  about  ten 
times  the  length  of  the  middle-piece  and  lacks  the  'broad  helix' 



Broken  end  of  tail  from  a  bull  spermatozoon,  showing  the  tuft  of  fibrils 
of  the  axial  filament,  and  the  helical  structure  of  the  tail  sheath; 
I 1  indicates  1  /x. 

(By  courtesy  of  Prof.  L.  H.  Bretschneider  and  Dr.  Woutera  van  Iterson) 

The  Two  Components  of  Semen  15 

but  has  instead  the  much  thinner  'tail  sheath'  or  'cortical  helix* 
which  terminates  a  short  distance  before  the  end  of  the  tail,  exposing 
the  terminal  portion  of  the  axial  filament,  that  is  the  end-piece.  In 
mammalian  spermatozoa,  the  tail  sheath  appears  as  a  helicoidally 
wound  cord;  when  the  tail  of  the  spermatozoon  is  broken,  one  can 
see,  protruding  from  the  cortical  sheath,  the  brush-like  fibrils  of  the 
axial  filament,  and  at  this  point  it  is  also  possible  to  distinguish  the 
helical  structure  of  the  tail  sheath  (Plate  II).  In  fowl  spermatozoa  on 
the  other  hand,  there  is  no  evidence  of  a  'cortical  helix',  and  the  axial 
filament  is  encased  in  an  amorphous  sheath  which  is  easily  dis- 
rupted by  distilled  water,  causing  the  axial  filament  to  fray  into 
fibrils.  In  addition  to  the  various  fibrous  cortical  systems,  the  sperm 
cell  of  many  species,  including  man  and  the  higher  mammals,  is 
protected  externally  by  a  lipid  layer  or  capsule  ('manteau  lipidique') 
evident  especially  around  the  tail,  and  composed  of  a  layer  of 


Seminal  plasma,  the  extracellular  fluid  which  provides  the  medium 
and  vehicle  for  spermatozoa,  originates  in  the  accessory  organs  of 
reproduction  and  varies  in  composition  according  to  species.  In 
lower  animals  it  may  be  so  scarce  that  the  emitted  semen  takes  the 
form  of  a  very  thick  lump  of  spermatozoa,  closely  packed  together. 
There  is  little  seminal  plasma  in  bird  semen  and  even  among  some 
of  the  mammals,  but  on  the  whole,  the  higher  mammals,  including 
man,  produce  a  relatively  dilute  semen  with  a  considerable  propor- 
tion of  seminal  plasma. 

Secretory  function  of  male  accessory  glands 

The  seminal  plasma  is  a  composite  mixture  of  fluids  secreted  by 
organs  which  in  the  higher  species  comprise  the  epididymides,  the 
seminal  ducts  or  vasa  deferentia,  ampullae,  prostate,  seminal  vesicles 
(or  seminal  glands),  Cowper's  glands  and  certain  other  glands 
located  in  the  wall  of  the  urethral  canal.  Until  a  little  while  ago,  the 
secretory  function  of  the  male  accessory  organs  remained  obscure 
chiefly  owing  to  lack  of  information  about  the  chemical  nature  of 
the  various  secretions.  More  recently,  however,  several  substances 


The  Biochemistry  of  Semen 

have  been  discovered  and  identified  in  the  accessory  secretions,  such 
as  citric  acid  by  Schersten  in  1929,  prostatic  phosphatase  by  Kutcher 
and  Wolbergs  in  1935,  fructose  by  Mann  in  1945,  phosphoryl- 
choline  by  Lundquist  in  1946,  ergothioneine  by  Leone  and  Mann, 
and  inositol  by  Mann  in  1951,  and  glycerylphosphorylcholine  by 

Rabbit  Rat 

Fig.  4.  Diagrammatic  outline  of  male  accessory  organs  to  illustrate  the 
localization  of  fructose  {shaded  areas). 

Am,  ampullae.  SV,  seminal  vesicle.  Pr,  prostate.  VP,  ventral  prostate. 

DLP,  dorsolateral  prostate.  Pp,  glandulae  paraprostaticae.  GV,  glandula 

vesicularis.  CG,  coagulating  gland. 

Diament,  Kahane  and  Levy  in  1952  (for  details  concerning  the 
secretory  function  of  male  accessory  organs  see:  Mann  and  Lutwak- 
Mann,  1951ft;  Lutwak-Mann,  1951). 

Owing  to  the  complex  nature  of  the  seminal  plasma  the  physio- 
logist or  biochemist  is  forced  to  adopt  a  distinct  approach  when 
investigating  any  one  of  the  accessory  gland  secretions.  There  are 
several  instances  where  male  accessory  organs  which,  though  pre- 
viously believed  on  the  basis  of  similar  embryonic  origin  or  related 



A,  prostate;  B,  seminal  vesicle;  C,  vas  deferens;  D,  Cowper's  gland;  E, 
caput  epididymidis;  F,  cauda  epididymidis;  G,  testis;  H,  bladder. 
Scale  in  inches. 

The  Two  Components  of  Semen  17 

morphological  structure,  to  be  anatomically  and  even  functionally 
'homologous',  were  later  shown  to  differ  greatly  in  their  chemical 
secretory  activity.  This  is  particularly  true  of  the  secretions  of  the 
prostate  and  the  seminal  vesicle,  two  organs  which  in  the  majority 
of  higher  species  provide  the  bulk  of  the  seminal  plasma;  their 
localization  within  the  reproductive  tract  of  several  species  is  illus- 
trated in  Plate  III  and  Fig.  4. 

The  prostatic  secretion 

This  differs  in  many  ways  from  other  secretions  of  the  mammalian 
body,  and  its  composition  shows  considerable  species  variations. 
Much  study  has  been  devoted  to  the  human  and  canine  prostatic 
fluids;  both  are  colourless  and  usually  slightly  acid,  about  pH  6-5 
(Huggins,  1947;  Zagami,  1940)  and  both  are  remarkable  for  the 
almost  complete  absence  of  reducing  sugar.  They  abound,  however, 
in  several  strong  proteolytic  enzymes;  the  human  prostatic  fluid 
contains  a  fibrinolysin  so  powerful,  that  2  ml.  of  prostatic  fluid  can 
liquefy  100  ml.  clotted  human  blood  in  18  hr.  at  37°;  dog  prostatic 
fluid  is  distinguished  by  its  ability  to  destroy  fibrinogen,  but  it  is 
relatively  inactive  towards  clotted  blood  (Huggins  and  Neal,  1942). 
The  prostate  secretes  a  diastase  (Karassik,  1927),  and  a  /3-glucuroni- 
dase  which  is  more  active  in  man  than  in  dog  (Talalay,  Fishman  and 
Huggins,  1946;  Huggins,  1947). 

The  prostatic  secretion  represents  the  main  source  of  citric  acid 
and  of  acid  phosphatase  for  whole  human  semen;  and  the  analysis 
of  these  two  constituents  provides  a  most  convenient  'chemical 
indicator  test'  for  the  assessment  of  the  functional  state  of  the 
human  prostate.  There  is  much  more  citric  acid  and  acid  phospha- 
tase in  the  human,  than  in  the  canine,  secretion;  thus,  the  citric  acid 
content  is  less  than  30  mg./lOO  ml.  in  dog,  as  against  480-2680 
mg./lOO  ml.  in  the  human  fluid;  acid  phosphatase  activity  in  dog 
corresponds  to  about  28  King-Armstrong  units/100  ml.  in  the 
'resting'  or  spontaneously  voided  prostatic  secretion,  and  104 
units/100  ml.  in  the  'stimulated'  secretion  obtained  by  parasym- 
pathetic stimulation,  whereas  the  prostatic  secretion  of  a  normal 
adult  man  may  contain  up  to  3950  units/1  ml.  (Gutman  and 
Gutman,  1941;  Huggins,  1947). 

The    concentration    of    osmotically-active    substances    in    the 

1 8  The  Biochemistry  of  Semen 

prostatic  fluids  of  man  and  dog  is  shown  in  Fig.  5.  In  the  human 
secretion  (Huggins,  Scott  and  Heinen,  1942),  the  average  values  for 
cations,  expressed  in  m-equiv./l.  water,  are:  sodium  156,  potassium 
30,  and  calcium  30;  for  anions:  citrate  156,  chloride  38,  bicarbonate 
8,  and  phosphate  1.  In  a  pilocarpine-stimulated  dog  prostatic  fluid 
(Huggins,  Masina,  Eichelberger  and  Wharton,  1939)  the  base  con- 
























Fig.  5.  Diagram  of  osmotically  active  substances  in  prostatic  fluid. 

(Huggins,  1947) 

sists  of  sodium  162,  and  potassium  5;  the  anions  chloride  156,  and 
bicarbonate  1-7  m-equiv./l.  In  man,  the  prostatic  secretion  also 
provides  the  main  source  of  calcium;  the  so-called  Niederland  re- 
action, which  depends  on  the  formation  of  characteristic  needle- 
shaped  crystals  in  human  semen  heated  with  dilute  sulphuric  acid, 
is  probably  due  to  calcium  sulphate  (Niederland,  1931,  1935; 
Ziemke,  1931). 
^  Among  the  chemical  peculiarities  of  the  prostate  gland  is  its 

The  Two  Components  of  Semen  19 

rather  high  content  of  zinc.  The  first  to  observe  this,  and  to  comment 
on  the  possible  role  of  zinc  in  reproduction,  was  Gabriel  Bertrand; 
in  the  two  analyses  of  human  prostate  carried  out  by  Bertrand  and 
Vladesco  (1921)  there  was  found  9-4  and  11-3  mg.  Zn  per  100  g. 
fresh  tissue,  or  49  1  and  53  1  mg.  Zn  per  100  g.  dry  weight.  More 
recently,  Mawson  and  Fischer  (1951,  1952,  1953)  in  Canada,  found 
that  the  mean  zinc  content  of  the  human  prostate  gland  was  68-2  mg. 
Zn/100  g.  dry  wt.,  which  is  in  considerable  excess  of  zinc  content 
in  human  liver,  muscle,  brain,  testis  or  blood.  These  investigators 
state  that  the  zinc  present  in  the  human  seminal  plasma  is  derived 
chiefly  from  the  prostatic  secretion. 

A  considerable  number  of  studies  have  been  carried  out  with  the 
rat  prostate  (Fig.  4).  In  the  rat,  there  is  a  distinct  anatomical  and 
functional  difference  between  the  so-called  ventral  prostate  which 
secretes  only  citric  acid,  and  the  dorso-lateral  prostate  which  pro- 
duces both  citric  acid  and  fructose  (Humphrey  and  Mann,  1948, 
1949).  In  the  dorso-lateral  prostate  itself,  however,  it  is  possible 
to  distinguish  three  smaller  regions,  the  dorsal  or  median  portion 
which  does  not  contribute  citric  acid,  and  two  lateral  lobes  which 
are  rich  in  citric  acid  (Price,  Mann  and  Lutwak-Mann,  1949).  It  is 
the  dorso-lateral  prostate,  and  more  specifically,  its  two  lateral  lobes 
which  contain  much  more  zinc  than  any  other  soft  tissue  of  the  rat, 
and  which  at  the  same  time  exhibit  carbonic  anhydrase  activity 
almost  equal  to  that  of  blood  (Mawson  and  Fischer,  1952);  whereas 
however,  the  carbonic  anhydrase  in  the  rat  lateral  prostate  accounts 
for  no  more  than  one-tenth  of  the  total  zinc  content,  in  blood 
erythrocytes  this  enzyme  is  well  known  to  correspond  closely  to 
the  bulk  of  the  zinc  content  (KeiUn  and  Mann,  1940). 

The  protein  content  of  the  prostatic  secretion  is  low,  less  than  1  % 
in  man,  and  a  certain  proportion  of  the  protein-like  material  present 
in  the  secretory  fluid  is  composed  of  'proteoses'  which  are  not  preci- 
pitated by  trichloroacetic  acid.  Another  feature  of  the  prostatic 
secretion  is  its  elevated  content  of  certain  free  amino  acids,  the 
presence  of  which  is  probably  the  outcome  of  a  combined  action 
of  proteolytic  and  transaminating  enzymes  in  the  glandular  tissue 
(Barron  and  Huggins,  1946a;  Awapara,  \952a,  b).  Human  prostatic 
adenoma  contains  in  100  g.  tissue  50  to  200  mg.  glutamic  acid  in 
addition  to  several  other  amino  acids.  The  average  content  of  amino 

20  The  Biochemistry  of  Semen 

acids  in  protein-free  filtrates  of  ground  prostatic  adenoma  or  dog 
prostate,  expressed  in  terms  of  millimoles/100  g.  tissue,  is  34  and 
39,  respectively.  The  ventral  lobe  of  the  rat  prostate  contains  in  a 
free  state  nearly  all  known  amino  acids,  and  in  addition  phosphoryl- 
ethanolamine,  taurine,  glutathione,  and  glutamine.  The  dorso-lateral 
lobe  on  the  other  hand,  in  contrast  to  the  ventral  prostate,  has  a 
much  lower  content  of  most  amino  acids  and  lacks  completely  iso- 
leucine  and  threonine;  it  may  be  added  here  that  it  also  responds 
differently  to  castration  and  to  hormones. 

The  seminal  vesicle  secretion  (Tables  2  and  3) 

In  several  species,  including  the  rat,  guinea-pig  and  bull,  the 
seminal  vesicles  alone  contribute  more  fluid  than  the  rest  of  the 

Table  2.  Some  characteristic  constituents  of  the  seminal  vesicle 
secretion  in  man,  bull,  boar,  rat  and  guinea-pig 

(In  each  species,  two  individuals  were  examined,  and  results  expressed 
as  averages  (mg./lOO  ml.);  the  human  vesicular  secretion  may  have  been 
contaminated  with  prostatic  fluid.  Ascorbic  acid  was  determined  as  dinitro- 
phenylhydrazone,  and  the  value  represents  the  sum  of  the  oxidized  and 
reduced  form.) 






Dry  weight 






Ascorbic  acid 






Citric  acid 












Inorganic  phosphorus 






Acid-soluble  phosphorus 












*  In  rat,  fructose  is  secreted  by  the  coagulating  gland,  an  organ  adjacent 
to  the  seminal  vesicle  (see  Fig.  4). 

accessory  glands  together.  Whereas  however,  in  the  rat  and  guinea- 
pig  the  seminal  vesicles  conform  to  the  pattern  of  true  thin-walled 
and  large  'vesicles',  the  bull  seminal  vesicles  are  more  correctly  des- 
cribed as  seminal  'glands',  with  multiple  lobes  of  glandular  tissue 
which  surrounds  a  system  of  ramified  secretory  ducts.  The  size  and 
storage  capacity  of  the  seminal  vesicles,  and  their  secretory  output, 

The  Two  Components  of  Semen  21 

are  subject  to  individual  variations,  which  are  particularly  conspicu- 
ous in  man.  But  the  storage  capacity  of  the  human  vesicles  is  small 
indeed  in  comparison  with  that  of  the  bull  or  boar.  In  certain  mam- 
mals such  as  the  dog  or  cat,  the  seminal  vesicles  are  altogether 
absent.  In  the  rabbit,  a  combined  anatomical  and  biochemical  study 
of  the  reproductive  system  has  shown  that  the  two  organs  known  as 
glandula  seminalis  and  glandula  vesicularis,  develop  from  the  same 
diverticulum  of  the  Wolffian  duct,  and  possess  a  common  urethral 
outlet,  so  that  both  these  glands  together  may  be  regarded  as  homo- 
logous to  the  seminal  vesicles  proper  of  other  mammals  (Davies  and 
Mann,  1947«,  b;  Mann,  1947). 

Table  3.  Composition  of  the  boar  seminal  vesicle  secretion 
(m^./lOO  ml.) 

(Analysis  of  670  ml.  seminal  vesicle  fluid  representing  the  pooled 
secretions  collected  from  three  boars;  sp.  g.  1046;  pH  7-2.) 

Dry  weight 



soluble  in  66%  ethanol 

18  635 

6  565 

12  070 

5  347 









Inorganic  phosphorus 
Inorganic  sulphur 
Total  nitrogen 
Non-protein  nitrogen 



1  548 







Total  anthrone-reactive 

Total  aminosugar 



3  053 

Total  sulphur 




Lactic  acid 


Citric  acid 


Total  phosphorus 
Acid-soluble  phosphorus 


22  The  Biochemistry  of  Semen 

Compared  with  the  prostatic  fluid,  the  seminal  vesicle  secretion 
is  usually  less  acid,  sometimes  distinctly  alkaline,  has  a  higher  dry 
weight  and  contains  more  potassium,  bicarbonate,  acid-soluble 
phosphate  and  protein;  the  latter  is  to  a  large  extent  precipitable  by 
trichloroacetic  acid  but  there  is  also  some  'proteose'  as  shown  for 
example,  by  the  study  of  the  seminal  vesicle  proteins  in  the  goby 
Gillichthys  mirabiUs  (Young  and  Fox,  1937).  But  the  most  remark- 
able feature  of  the  seminal  vesicle  secretion  is  its  unusually  high 
content  of  reducing  substances. 

The  normal  seminal  vesicle  secretion  is  usually  slightly  yellowish 
but  occasionally,  especially  in  man  and  bull,  it  can  be  deeply  pig- 
mented. The  yellow  pigmentation  is  probably  of  composite  origin 
but  much  of  it  is  due  to  flavins  which  cause  the  vesicular  secretion  and 
seminal  plasma  to  fluoresce  strongly  in  ultraviolet  light.  Brochart 
(1952)  observed  that  when  strongly  yellow  coloured  samples  of  bull 
seminal  plasma  are  exposed  to  sunHght,  the  colour  tends  to  dis- 
appear within  a  short  time  and  lumiflavin  is  formed.  Leone  (1953) 
has  shown  that  at  least  part  of  the  flavin  content  of  the  bull  seminal 
vesicle  secretion  is  due  to  adenine-isoalloxazine  dinucleotide, 
associated  with  xanthine  oxidase.  The  highest  content  of  total  flavin 
which  I  was  able  to  record  in  the  bull  seminal  vesicle  secretion,  was 
750  /^g./lOO  ml.;  in  eight  samples  of  bull  seminal  plasma  there  was 
from  47  yf^g./lOO  ml.  (in  an  almost  colourless  specimen)  to  480 
^g./lOO  ml.  flavin  (in  a  particularly  deeply  pigmented  specimen). 
There  can  be  little  doubt  that  the  flavin  associated  with  the  strongly 
yellow-coloured  specimens  of  bull  semen  is  due  principally  to  the 
seminal  plasma,  and  not  to  the  spermatozoa.  The  sperm  cells  them- 
selves, however,  contain  also  some  flavin.  In  washed  bull  sperma- 
tozoa, there  is  some  30  [ig.  riboflavin/g.  dry  weight  (Lardy  and 
Phillips,  1941c),  and  in  whole  bull  semen,  particularly  in  the  less 
coloured  samples,  a  substantial  portion  of  flavin  may  be  derived 
from  the  spermatozoa  (VanDemark  and  Salisbury,  1944). 

In  addition  to  the  yellow  pigment,  the  seminal  plasma  sometimes 
contains  a  brownish  haematin  pigment;  this  occurs  in  cases  of 
'chronic  haemospermia',  a  condition  occasionally  met  with  in  man 
and  attributed  to  haemorrhagic  changes  in  the  seminal  vesicles 
(McDonald,  1946). 

Potassium  in  a  high  concentration  occurs  in  the  vesicular  secretion 

The  Two  Components  of  Semen  23 

of  several  species,  including  man  (20  mM),  bull  (100  mM)  and 
boar  (300  mM).  In  the  latter,  the  ionic  equilibrium  on  the  cationic 
side  is  set  up  chiefly  by  potassium,  with  citric  acid  as  the  main  anion; 
the  concentration  of  sodium  is  much  less  than  that  of  potassium; 
chlorides  are  conspicuous  by  their  almost  total  absence,  a  phe- 
nomenon infrequently  encountered  in  other  normal  body  fluids 
(Table  3).  Another  unusual  feature  of  the  boar  vesicular  secretion  is 
its  high  content  of  inositol  which  varies  from  2  to  3%  and  accounts 
for  something  like  one-third  of  the  total  dialysable  material;  inositol 
together  with  citrate,  contributes  substantially  to  the  osmotic  pres- 
sure of  the  vesicular  secretion  (Mann,  1954). 

The  reducing  power  of  the  vesicular  secretion  is  one  of  its  most 
characteristic  chemical  properties.  Two  kinds  of  reducing  sub- 
stances are  present.  One  group  is  made  up  of  substances  which 
are  capable  of  reducing  silver  nitrate,  iodine  and  2  :  6-dichlorophenol 
indophenol  in  the  cold.  They  are  always  present  in  the  protein-free 
filtrate  from  the  vesicular  secretion  and  seminal  plasma  but 
their  chemical  nature  varies  from  one  species  to  another.  At 
one  time,  the  reducing  property  was  generally  attributed  to 
ascorbic  acid,  in  the  secretions  of  the  guinea-pig  (Zimmet  and 
Sauser-Hall,  1936;  Zimmet,  1939),  bull  (Phillips,  Lardy,  Heiser  and 
Ruppel,  1940;  Jacquet,  Cassou,  Plessis  and  Briere,  1950)  and  man 
(Nespor,  1939;  Berg,  Huggins  and  Hodges,  1941).  According  to 
Phillips  and  his  co-workers,  bulls  of  high  fertility  produce  semen 
containing  3  to  8  mg.  ascorbic  acid  per  100  ml.,  whereas  low-fertility 
bulls  may  have  less  than  2  mg./lOO  ml.;  these  workers  have  also 
claimed  that  in  certain  bulls  it  was  possible  to  enhance  the  fertility 
by  parenteral  administration  of  ascorbic  acid.  More  recent  studies, 
however,  based  upon  chemical  methods  of  purification  and  identi- 
fication, have  proved  that  ascorbic  acid  rarely  accounts  for  the  total 
reducing  power  of  semen  towards  dichlorophenol  indophenol.  In 
the  boar  especially,  ascorbic  acid  has  been  shown  by  Mann  and 
Leone  (1953)  to  account  but  for  a  small  fraction  of  seminal  reducing 
power  and  the  bulk  of  the  reducing  material  was  found  to  consist 
or  ergothioneine,  a  substance  which  owes  its  reducing  power  to  a 
sulphydryl  group  (Leone  and  Mann,  1951;  Mann  and  Leone,  1953). 
The  properties  and  functions  of  ergothioneine  will  be  discussed 
later  (p.  174). 

24  The  Biochemistry  of  Semen 

The  presence  of  the  other  kind  of  reducing  substances  can  be 
detected  in  protein-free  filtrates  from  semen  and  seminal  vesicle 
fluid,  by  heating  with  sugar  reagents,  such  as  for  example,  cupric 
hydroxide.  In  this  category  belongs  fructose,  the  physiological  sugar 
of  semen. 

Huggins  and  Johnson  (1933)  were  the  first  to  observe  that  the 
reducing  sugar  of  human  semen  is  derived  from  the  secretion  of  the 
seminal  vesicles  but  is  absent  in  the  prostate.  Similar  findings  were 
made  with  the  bull  (Bernstein,  1937),  boar  (McKenzie,  Miller  and 
Bauguess,  1938)  and  ram  (Moore  and  Mayer,  1941).  The  identifi- 
cation of  the  seminal  sugar  as  fructose  (Mann,  1946a,  b,  c)  opened 
the  way  for  detailed  studies  of  the  fructose-generating  capacity  of 
the  accessory  tissues  (Fig.  4).  It  was  shown  that  in  several  species 
fructose  is  secreted  either  by  the  seminal  vesicles  or  by  functionally 
related  organs  (Mann,  1946c;  1947;  l94Sa,  b).  This  made  it  possible 
to  use  the  chemical  assay  of  fructose  in  semen  as  an  indicator  of  the 
relative  contribution  made  by  the  seminal  vesicles  towards  the  make- 
up of  the  whole  semen.  It  must  be  pointed  out,  however,  that  a 
certain  small  amount  of  fructose  is  also  produced  by  the  ampullar 
glands  and  in  some  species,  by  certain  other  accessory  organs  (see 
p.  138). 

The  physiological  function  of  the  seminal  plasma 

From  time  to  time  doubt  is  expressed  as  to  whether  the  individual 
accessory  gland  secretions  or  even  the  entire  seminal  plasma,  have 
any  essential  role  to  fulfil  in  the  process  of  reproduction;  the  more 
so,  since  in  some  anmials  such  as  the  guinea-pig  or  rabbit,  it  is 
possible  to  induce  pregnancy  by  the  artificial  insemination  of  epi- 
didymal  spermatozoa.  It  is  however,  arguable  as  to  how  much  sig- 
nificance may  be  ascribed  to  such  experiments,  and  it  is  certain  that 
the  natural  mating  process  could  scarcely  be  expected  to  function 
smoothly  and  efficiently  without  the  provision  of  seminal  plasma 
as  a  normal  diluent  and  vehicle  for  the  thick  mass  of  closely  packed 
epididymal  spermatozoa;  no  more  could  the  blood  corpuscles  act 
as  oxygen  carriers  in  vivo,  without  the  blood  plasma. 

Furthermore,  the  seminal  plasma  exerts  a  distinct  stimulating 
effect  on  sperm  motility.  In  part,  this  is  due  simply  to  the  'dilution 
effect',  a  phenomenon  which  is  described  fully  elsewhere  (see  p.  73). 

The  Two  Components  of  Semen  25 

To  a  considerable  extent,  however,  the  activation  by  seminal  plasma 
has  been  shown  to  be  linked  with  the  occurrence  of  specific  sub- 
stances in  the  different  accessory  gland  secretions.  In  certain  insects, 
Bombyx  mori  for  example,  the  spermatozoa  are  believed  to  acquire 
their  full  fertilizing  capacity  only  after  activation  by  the  secretion 
of  the  lower  portion  of  the  male  tract,  the  so-called  glandula  pros- 
tatica  (Wigglesworth,  1950).  Many  investigators  have  studied  the 
activating  influence  of  the  prostatic  secretion  on  mammalian  sper- 
matozoa (Steinach,  1894;  Hirokawa,  1909;  Ivanov,  1929;  Sergijewski 
and  Bachromejew,  1930),  and  in  some  instances,  e.g.  the  dog,  have 
claimed  it  to  be  species-specific  (Ivanov  and  Kassavina,  1946),  By 
no  means  all  of  these  experiments,  however,  have  been  carried  out 
under  satisfactorily  controlled  conditions  and,  as  Huggins  (1945) 
thinks,  in  many  cases  it  is  impossible  to  exclude  the  action  of  non- 
specific factors  such  as  acceleration  of  sperm  motility  by  certain  ions 
or  by  changes  in  the  gas  tension. 

Much  discrimination  is  equally  needed  in  appraising  certain 
results  obtained  with  the  epididymal  secretion.  Here,  however,  the 
problem  involves  not  so  much  the  'activation',  as  the  'ripening'  and 
'life-prolonging',  effects  on  the  spermatozoa  (Tournade  and  Mer- 
land,  1913;  Stigler,  1918;  Braus  and  Redenz,  1924;  Hammond  and 
Asdell,  1926;  Young,  1929,  1930;  Young  and  Simeone,  1930;  Lanz, 
1931,  1936;  Gunn,  1936;  Gunn,  Sanders  and  Granger,  1942).  Most 
investigators  agree  that  in  some  special  way  the  epididymis  is 
adapted  for  the  long-term  storage  of  spermatozoa;  but  whereas  some 
like  Braus  and  Redenz  (1924),  attribute  to  the  epididymal  secretion 
a  specific  role  in  the  'ripening'  process,  others  deny  it  such  a  func- 
tion. In  the  seminal  vesicle  fluid,  we  seem  to  be  confronted  with  an 
activating  effect  on  spermatozoa  different  from  that  exerted  by  the 
prostatic  and  epididymal  secretions,  one  which  is  specifically  bound 
up  with  the  presence  therein  of  fructose  which  provides  a  source  of 
nutrient  material  for  the  sperm  cells. 

Another  facet  of  the  physiological  function  of  the  seminal  plasma 
concerns  certain  characteristic  pharmacological  effects  of  the  acces- 
sory gland  secretions  and  the  role  of  seminal  plasma  in  the  remark- 
able process  of  semen  coagulation  and  liquefaction. 

26  The  Biochemistry  of  Semen 

Prostaglandin y  vesigiandin,  and  certain  other  pharmacodynamically 
active  substances 

Among  the  more  striking  pharmacological  effects  of  seminal 
plasma  are  a  depressor  action  on  blood  vessels,  and  a  stimulation 
of  isolated  smooth-muscle  organs  such  as  the  uterus  and  the  intes- 
tines. Both  these  effects  which  have  been  studied  in  great  detail  by 
von  Euler  (1934«,  b\  1935,  1937,  1939,  1949)  and  Goldblatt  (1933, 
1935^?)  are  due  in  all  probability  not  to  a  single  substance  but  to  the 
combined  action  of  several  constituents  of  seminal  plasma,  including 
choline  and  two  substances  which  Euler  has  named  'prostaglandin* 
and  'vesigiandin'.  So  far,  only  prostaglandin  has  been  purified. 
The  purest  preparation  obtained  from  ram  prostate  glands  has  been 
found  by  Bergstrom  (1949)  to  be  nitrogen-free,  and  to  contain  an 
unsaturated  acidic  substance  which  absorbs  strongly  ultraviolet  light 
with  a  maximum  at  280  m//.  When  assayed  on  the  isolated  intestine 
of  rabbit,  1/^g.  of  this  substance  exhibited  the  same  activity  as  the 
crude  extract  from  500  mg.  prostatic  tissue.  Assuming  that  the  sub- 
stance is  pure,  the  total  content  of  prostaglandin  in  100  kg.  prostate 
glands  must  be  of  the  order  of  25-50  mg.  To  prepare  this  quantity, 
one  would  require  the  glands  from  20,000  rams. 

The  physiological  significance  of  prostaglandin  and  vesigiandin  in 
reproduction  processes  is  unknown  but  it  has  been  suggested  that 
they  represent  some  sort  of  'automatic  regulator'  which  controls 
the  voiding  of  the  secretions  from  the  prostate  and  the  seminal 
vesicle,  respectively.  The  idea  of  chemical  stimulation  by  secretory 
products  is  based  among  others,  on  observations  that  the  emptying 
of  the  prostate  and  seminal  vesicle  leads  to  a  decreased  ability  of 
these  glands  to  contract  which  persists  until  enough  of  freshly 
formed  secretion  has  accumulated. 

A  pharmacodynamic  influence  of  the  seminal  plasma  upon  some 
parts  of  the  female  reproductive  tract  has  also  been  envisaged  but 
the  evidence  is  derived  mainly  from  experiments  on  isolated  organs. 
It  remains  questionable  whether  any  of  the  effects  exerted  by  the 
seminal  plasma  on  the  uterus  and  oviduct  in  vitro,  also  occur  in  the 
female  reproductive  tract  in  vivo  (Barnes,  1939;  Asplund,  1947). 

Kurzrok  and  Lieb  (1931)  found  on  adding  1  ml.  of  human  seminal 
fluid  to  a  strip  of  human  uterus  suspended  in  a  100  ml.  bath,  either 

The  Two  Components  of  Semen  27 

an  increase  or  a  decrease  in  spontaneous  contractions.  Cockrill, 
Miller  and  Kurzrok  (1935)  observed  that  those  specimens  of  human 
semen  which  were  capable  of  enhancing  uterine  contraction,  caused 
an  inhibition  after  having  been  exposed  for  half  an  hour  to  pH  10; 
at  pH  11  all  specimens  became  inactive.  Moreover,  the  effect  was 
potentiated  by  eserine  and  suppressed  by  atropine.  The  observed 
action  corresponded  to  that  of  about  100  ^g.  acetylcholine  per  ml. 
semen.  About  that  time,  the  occurrence  of  a  powerful  oxytocic 
substance  in  the  human  seminal  plasma  was  demonstrated  by  Gold- 
blatt  {\92>5b)  who  also  found  that  the  activity  was  destroyed  by 
short  boiling  of  the  seminal  plasma  with  either  OlN-NaOH  or 
OlN-HCl.  When  assayed  on  the  guinea-pig's  uterus,  1  ml.  of  human 
seminal  plasma  showed  approximately  the  same  oxytocic  activity 
as  0-4-0-6  mg.  of  histamine.  Euler  (1937)  believes  that  the  oxytocic 
activity  is  due  to  prostaglandin  which  in  his  experiments  stimulated 
strips  of  human  uterus  and  also  isolated  uterus  as  well  as  uterine 
strips  from  several  species  including  the  cow,  rabbit,  guinea-pig  and 
rat.  Asplund  (1947)  determined  the  total  content  of  'contractive  sub- 
stance' in  155  specimens  of  human  semen  which  he  assayed  on  the 
rabbit  intestine  in  vitro,  with  purified  prostaglandin  as  the  standard. 
He  came  to  the  conclusion  that  the  effect  of  semen  on  the  isolated 
intestine  must  be  attributed  to  a  combined  action  of  prostaglandin, 
choline,  and  at  least  one  other  substance  which  produces  a  very 
rapid  increase  in  tonus  and  is  unaffected  by  atropine.  There  was  no 
correlation  between  the  total  content  of  'contractive  substance'  in 
seminal  plasma  and  the  motility  and  viability  of  spermatozoa. 

In  addition  to  the  remarkable  vaso-dilation  and  contraction  of 
plain  muscle,  the  seminal  plasma  and  the  accessory  gland  secretions 
exhibit  a  characteristic  pressor  activity  towards  blood  vessels.  In 
1906  Jappelli  and  Scafa  found  that  parenterally  administered  extracts 
of  the  canine  prostate  produced  an  increased  blood  pressure  in  the 
dog  and  stimulated  the  respiration.  A  similar  effect  was  observed  by 
Thaon  (1907)  after  the  intravenous  injection  of  prostatic  extracts  into 
rabbits  but  in  his  experiments  the  rise  in  blood  pressure  was  usually 
followed  by  a  fall.  There  are  indications  that  the  pressor  action  of 
the  prostatic  extracts  is  due  to  adrenaline-like  substances  demon- 
strated in  semen  and  accessory  glands  by  CoUip  (1929),  v.  Euler 
(1934Z>),  Bacq  and  Fischer  (1947),  and  Brochart  (1948fl)  (see  p.  181). 

28  The  Biochemistry  of  Semen 

Coagulation  and  liquefaction 

Semen  is  ejaculated  in  a  liquid  or  semi-liquid  form.  In  some  animal 
species  such  as  the  bull  and  the  dog,  it  remains  liquid,  but  in  others 
it  tends  to  gelate  or  coagulate  shortly  after  ejaculation.  Human 
semen  clots  immediately  after  ejaculation  only  to  liquefy  again  a 
little  later;  until  that  happens  the  spermatozoa  do  not  become  fully 
motile.  For  this  reason  the  examination  of  human  spermatozoa 
should  be  postponed  until  at  least  twenty  minutes  after  the  emission 
of  semen  (McLeod,  1946<2;  Sunmons,  1946).  Quite  fresh  boar  semen 
usually  contains  only  small  lumps  of  gelatinous  material  somewhat 
resembling  tapioca;  on  standing,  however,  the  lumps  increase  and 
merge  into  a  semi-solid  gelatinous  mass  which  may  take  up  half  or 
more  of  the  entire  ejaculate.  Gelation  of  semen  can  also  be  observed 
in  the  stallion.  Even  more  striking  is  the  clotting  phenomenon  in 
the  rodents.  The  major  part  of  a  rabbit  ejaculate  collected  by  means 
of  an  artificial  vagina  often  consists  of  a  colourless  transparent  gel. 
In  rats  and  guinea-pigs,  semen  coagulation  leads  to  the  formation 
after  mating,  of  the  so-called  bouchon  vaginal  or  vaginal  plug,  which 
probably  prevents  the  back-flow  of  semen  from  the  vagina  and 
assists  the  passage  of  spermatozoa  through  the  cervix  into  the  uterus. 
In  a  study  of  sperm  transport  in  the  rat,  Blandau  (1945)  has  shown 
that  the  ejaculate  fails  to  pass  through  the  uterine  cervix  if  the 
coagulation  of  semen  is  aboUshed  by  ligation  of  the  seminal  vesicle 
and  coagulating  gland  ducts.  The  copulatory  plug  has  also  been 
described  in  Insectivora  (mole  and  hedgehog),  Chiroptera  {Rhinolo- 
phidae  and  Vespertilionidae)  and  in  Marsupialia  (Camus  and  Gley, 
1899;  Courrier,  1927;  Eadie,  1939,  1948a,  b;  Engle,  1926;  Rollinat 
and  Troussart,  1897;  Stockard  and  Papanicolaou,  1919).  In  most 
animals  the  vaginal  plug  is  due  to  the  clotting  of  the  semen  itself  but 
in  some,  namely  in  the  opossum  (Hartman,  1924)  and  in  the  bat 
Vesperuga  noctula  (Courrier,  1925;  Grosser,  1903),  its  occurrence 
involves  the  coagulation  of  female  secretions  by  the  seminal  plasma. 
In  the  honey  bee,  the  escape  of  semen  from  the  female  reproductive 
tract  is  prevented  by  the  so-called  mucus  plug;  this  is  formed  by  the 
material  ejected  from  the  mucus  glands  of  the  drone  towards  the  end 
of  ejaculation  (Laidlaw,  1944). 

In  most  species  the  substrate  for  gel  formation  consists  of  protein- 

The  Two  Components  of  Semen  29 

like  material  secreted  by  the  seminal  vesicles  (Bergmann  and  Leuc- 
kart,  1855;  Bischoff,  1852;  Hensen,  1876;  Landwehr,  1880;  Lataste, 
1888;  Pittard,  1852;  Stockard  and  Papanicolaou,  1919),  the  enzyme 
however,  responsible  for  the  coagulation  is  absent  in  the  vesicles 
themselves  and  comes  into  contact  with  the  protein  substrate  only 
in  the  course  of  ejaculation.  The  first  to  recognize  the  enzymic 
nature  of  the  coagulating  agent  were  Camus  and  Gley  (1896, 
1899,  1907,  1921,  1922)  who  named  it  'vesiculase'.  It  was  shown  by 
Walker  (1910)  that  the  source  of  the  coagulating  enzyme  in  small 
rodents  is  the  so-called  'coagulating  gland',  which  lies  adjacent  to 
the  seminal  vesicle.  The  clotting  power  of  the  secretion  from  the 
coagulating  gland  is  such  that  1  part  is  sufficient  to  clot  20,000  parts 
of  the  seminal  vesicle  secretion.  This  quantitative  relationship  can 
be  demonstrated  in  vitro  by  collecting  the  two  fluids  separately  and 
then  mixing  them  after  suitable  dilution  (Moore  and  Gallagher, 

The  liquefaction  of  human  semen  which  follows  its  coagulation,  is 
also  an  enzymic  process.  Fibrinolysin  and  fibrinogenase,  two  pro- 
teolytic enzymes  of  semen  (see  p.  1 14),  are  believed  to  play  a  part  in 
this  process. 


Chemical  and  Physical  Properties 
of  Whole  Ejaculated  Semen 

Species  and  individual  variations  in  the  composition  of  semen.  Pre- 
sperm,  sperm-containing,  and  post-sperm  fractions  in  the  ejaculate. 
Criteria  for  the  rating  of  semen  quality.  Optical  and  electrical  properties 
of  semen.  Viscosity,  specific  gravity,  osmotic  pressure,  and  ionic  equi- 
librium. Hydrogen  ion  concentration  and  buffering  capacity.  Metabohsm 
of  semen  and  its  relation  to  sperm  density  and  motility;  glycolysis; 
methylene-blue  reduction  test;  respiration. 

Species  and  individual  variations  in  the  composition  of  semen 
A  CHARACTERISTIC  feature  of  whole  semen  is  the  variability  of  its 
composition  not  only  among  different  species  but  also  between  in- 
dividuals of  the  same  species  (Table  4).  Somewhat  less  pronounced 
but  still  significant  are  the  variations  in  the  concentration  of  some 
of  the  semen  constituents  in  the  same  individual  (Table  5).  All  this 
is  not  altogether  unexpected  since  both  the  spermatogenic  activity 
of  the  testes  and  the  secretory  function  of  the  male  accessory  organs 
are  subject  to  considerable  physiological  fluctuations  of  hormonal 
origin  and  are  influenced  by  factors  such  as  light,  temperature, 
season,  state  of  nutrition  etc.  The  variability  of  semen  composition 
is  the  reason  why  repeated  examination  of  whole  semen,  even  if 
restricted  to  a  single  experimental  subject,  and  carried  out  under 
identical  experimental  conditions,  need  not  yield  the  same  quanti- 
tative results,  as  might,  for  example,  an  analogous  series  of  blood 
analyses  in  the  same  individual.  Therefore,  an  accurate  estimate  of 
semen  quality  in  any  one  individual  cannot  be  formed  on  the  basis 
of  a  single  analysis  and  involves  several  examinations. 

In  Table  5  are  shown  individual  fructose  variations  in  the  semen 
of  man,  bull,  ram  and  rabbit.  The  analytical  results  are  given  in 
terms  of  concentration  (mg./lOO  ml.  semen)  and  of  absolute  content 


Chemical  and  Physical  Properties  of  Semen         3 1 

(mg, /ejaculate).  This  method  of  expressing  the  findings  of  semen 
analysis  is  very  useful  particularly  in  the  evaluation  of  pathological 
abnormalities  in  the  composition  of  semen.  The  data  in  Table  5, 
especially  those  relating  to  rabbit  semen,  show  that  the  variations 
in  the  absolute  content  of  fructose  per  single  ejaculate  are  much 
less  than  those  in  concentration;  the  reason  being  that  a  rabbit 


-      3-0 



/I               \ 


/  \ 



-     2-5 


/           i       '     Vi 




_   8 


-     2-0 



/       \           '/ 


/  \  * 

/    \* 


1    6 

-     1-5 






-      1-0^ 


1  1^ 
,  / 



-     0-5 

1      1      1      1 


1-5   % 

012  34S6  789         10 

Time  (weeks) 

12       13        14v 

Fig.   6.    Relation  between  volume  (( 

and  content  of  fructose  (0 — 
and  citric  acid  {% •)/«  rabbit  semen 

(Mann  &  Parsons,  1950) 

•)   of  ejaculate 


ejaculate  is  usually  made  up  of  two  main  portions,  gel  and  fluid,  of 
which  the  latter  is  relatively  constant  whereas  the  quantity  of  gel 
is  subject  to  considerable  fluctuation.  Fructose,  however,  is  con- 
fined chiefly  to  the  fluid  portion  and  consequently,  its  absolute 
content  in  semen  varies  little.  On  the  other  hand,  citric  acid,  another 

Table  4.  Species  differences  in  the  chemical  composition  of  semen 

(Results  are  average  values  (range  in  brackets)  expressed  in  mg./lOO  ml. 
except  for  COg  content  (ml. /1 00  ml.);  for  information  on  the  composition 
of  dog  and  cock  semen  see  appendices  (6)  and  (7).) 






Dry  weight 







Chloride  (CI) 


















































Total  nitrogen 



















Uric  acid 





















Lactic  acid 






Citric  acid 










Total  phosphorus         1 1 2 











Lipid  phosphorus            6 











(1)  Human  semen.  Data  on  dry  weight,  total  nitrogen,  and  electrolytes 
(excluding  inorganic  phosphate):  Huggins,  Scott  and  Heinen  (1942);  COg: 
Sheldovsky  et  al.  (1940);  urea:  Goldblatt  (1935^);  Lactic  acid:  Lundquist 
(1949Z>).  Remaining  data  are  our  own;  the  results  on  non-protein  nitrogen, 
fructose,  and  citric  acid  content  are  based  on  analysis  of  semen  from  ten 
individuals.  The  ash  content  of  human  semen  is  about  0-9%  (Slovtzov, 
1902,  1916);  the  ash  contains  3%  sulphur  (Albu,  1908).  According  to 
Infantellina  (1945)  human  semen  contains  30  mg./lOO  ml.  glutathione. 
The  values  reported  for  ascorbic  acid  are  12-8  mg./lOO  ml.  (Berg,  Huggins 
and  Hodges,  1941)  and  2-6-3-4  mg./lOO  ml.  (Nespor,  1939);  using  Roe's 
method,  we  found  10-2  and  12-4  mg./lOO  ml.  in  two  specimens. 

(2)  Bull  semen.  Data  on  electrolytes  (except  magnesium  and  inorganic 
phosphate):  Bernstein  (1933);  COg:  Shergin  (1935);  remaining  data  are  our 
own,  based  on  analysis  of  ejaculates  from  ten  bulls  (average  volume  of 
ejaculate  4-5  ml.;  sperm  concentration  985,000/jul.).  Other  constituents  of 
bull  semen  include  (mg./lOO  ml.):  thiamine  0-028-0-152,  riboflavin  0-152- 
0-306,  pantothenic  acid  0-230-0-466,  and  niacin  0-248-0-554  (VanDemark 
and  Sahsbury,  1944).  Ehlers  et  al.  (1953)  reported  the  following  mean 
values  based  on  the  analysis  of  663  samples  of  semen  (mg./lOO  ml.): 
fructose  552  (S.D.  169),  citric  acid  724  (S.D.  192).  For  additional  data  see 
Rothschild  and  Barnes  (1954). 

(3)  Ram  semen.  With  the  exception  of  the  CO2  content  (Shergin,  1935), 
the  data  are  our  own,  based  on  analysis  of  material  pooled  from  ten 
ejaculates  (average  volume  of  single  ejaculate  1-2  ml.;  2,940,000  sperm/jitl.). 

(4)  Boar  semen.  Data  on  dry  weight,  electrolytes  and  total  nitrogen: 
McKenzie,  MUler  and  Bauguess  (1938);  remaining  data  our  own.  Addi- 
tional information  in  Table  7. 

(5)  Stallion  semen.  Data  on  dry  weight,  electrolytes,  total  phosphorus 
and  CO2  content  from  Slovtzov  (1916),  Bernstein  (1933),  Shergin  (1935) 
and  Milovanov  (1938);  remaining  data  our  own. 

(6)  Dog  semen.  Much  work  has  been  done  on  the  dog  prostatic  secretion 
(see  text).  Whole  semen  has  been  analysed  by  Slovtzov  (1916)  and  the 
following  composition  found  (mg./lOO  ml.):  dry  weight  2450,  albumin, 
globulin  and  nucleoprotein  866,  mucoprotein  57,  and  lipid  182.  Electro- 
lytes have  been  examined  by  Bernstein  (1933);  dog  semen  is  distinguished 
by  a  high  content  of  chloride  (620-657  mg./lOO  ml.).  Fructose  and  citric 
acid  occur  only  in  traces. 

(7)  Cock  semen.  In  material  from  six  pooled  ejaculates,  5,100,000 
sperm//il.,  we  found  (mg./lOO  ml.):  57  total  anthrone-reactive  carbo- 
hydrate, of  which  4  was  fructose  and  41  glucose  (determined  by  glucose 
oxidase);  44  total  phosphorus,  of  which  27  was  acid  soluble;  2  ammonia. 
In  six  individual  specimens  of  cock  semen  we  found  from  7-7  to  81 
mg./lOO  ml.  glucose  but  never  more  than  4  mg./lOO  ml.  fructose. 



The  Biochemistry  of  Semen 

Table  5.  Individual  variations  in  the  level  of  fructose  in  semen 
(Ejaculates  collected  at  weekly  intervals.) 








100  ml.) 


100  ml.) 


100  ml.) 


100  ml.) 
















































































































characteristic  chemical  component  of  rabbit  semen,  is  found  largely 
in  the  gel,  and  its  varying  content  reflects  the  variability  of  the 
latter  (Fig.  6). 

Frequency  of  ejaculation  also  affects  the  composition  of  individual 
samples  of  semen.  Table  6  shows  the  results  of  a  so-called  exhaustion 
effect  on  the  semen  of  a  bull  (Mann,  1948«);  in  this  experiment,  eight 
ejaculates  were  collected  from  the  same  animal  within  63  min.,  at 
7-10  min.  intervals.  As  a  result  of  the  multiple  collections,  the  sperm 
density  fell  from  1,664,000  cells///l.  in  the  first,  to  98,000  cells/^ul. 
in  the  last,  ejaculate.  This  decrease,  however,  was  not  accompanied 
by  a  corresponding  diminution  of  fructose  concentration,  which  was 
much  the  same  in  the  first  (760  mg./lOO  ml.)  and  last  (690  mg./lOO 
ml.)  ejaculate.  But  it  must  be  remembered  that  the  seminal  vesicles 
of  a  bull  differ  from  those  of  man  and  other  mammals  by  their 
exceptional  capacity:  in  some  bulls  it  is  possible  to  recover  up  to 
50  ml.  of  secretory  fluid  from  the  seminal  vesicles,  enough  to  provide 
at  least  a  dozen  fructose-rich  ejaculates. 

Chemical  and  Physical  Properties  of  Semen         35 

Table  6.  Ejfect  of  frequency  of  collection  on  sperm  density  and  on 

concentration  of  fructose  and  lactic  acid  in  fresh  bull  semen 

(Mann,  1948a) 


Time  of 



Volume  of 



Sperm  density 

(thousands/ 1/d. 



(mg./lOO  ml. 


Lactic  acid 

(mg./lOO  ml. 


















































Pre-sperm,  sperm-containing,  and  post-sperm  fractions  in  the  ejaculate 
To  assess  correctly  the  composition  of  individual  specimens  of 
semen  it  is  essential  to  bear  in  mind  the  fact  that  in  some  species 
(e.g.  man,  boar,  stallion)  the  different  portions  of  semen  follow 
one  another  in  a  definite  order  of  sequence.  This  has  been  demon- 
strated by  the  so-called  split-ejaculate  method  which  depends  on  the 
collection  and  analysis  of  separate  fractions  of  the  same  ejaculate 
according  to  the  time  of  delivery  from  the  urethra.  In  man,  the 
ejaculation  is  initiated  by  the  secretion  of  Cowper's  glands,  the  pros- 
tatic secretion  is  delivered  next,  to  be  followed  by  the  sperm  and  the 
vesicular  secretion  (Broesike,  1912;  Huggins  and  Johnson,  1933; 
MacLeod  and  Hotchkiss,  1942;  Lundquist,  19496;  Pryde,  1950); 
according  to  Lundquist,  in  man  the  prostatic  secretion  contributes 
from  13  to  32%  and  the  vesicular  secretion  from  46  to  80%  of  the 
whole  ejaculate. 

In  the  boar,  an  animal  with  a  protracted  period  of  ejaculation, 
the  semen  consists  of  two  portions,  gel  and  liquid.  McKenzie  et  al. 
(1938)  calculated  that  15-20%  of  the  liquid  portion  is  derived  from 
the  seminal  vesicles,  2-5%  from  the  epididymis,  10-25%  from 
Cowper's  gland,  and  the  rest  is  made  up  by  the  urethral  glands 
secretion.  We  have  made  a  similar  investigation  (Mann  and  Glover, 
1954)  using  estimations  of  sperm  concentration  in  ejaculated  and 
epididymal  semen  for  the  assessment  of  the  epididymal  contribution, 
and  the  chemical  determinations  of  fructose,  citric  acid  and  ergo- 
thioneine  in  the  ejaculated  semen  and  in  the  vesicular  secretion  as 

36  The  Biochemistry  of  Semen 

a  means  for  the  evaluation  of  the  contribution  of  the  seminal  vesicles. 
From  the  results  of  this  study  (Table  7)  it  can  be  seen  that  in 
ejaculated  semen  (liquid  portion),  the  sperm  concentration  was  3% 

Table  7.  Composition  of  ejaculated  semen,  epididymal  semen,  and 
seminal  vesicle  secretion  of  the  same  boar 

(Four  ejaculates  were  collected  from  the  same  boar,  at  weekly  intervals. 
The  boar  was  then  killed,  and  the  epididymal  semen  and  vesicular  secretion 
collected.  For  chemical  analysis,  the  fluid  portions  were  used,  separated 
from  sperm  and  gel  by  centrifugation.  In  the  case  of  ejaculated  semen,  the 
means  (and  standard  deviation)  are  given  per  single  ejaculate.) 





from  the 





Total  volume  (ml.) 

375  (±24) 



Sperm  concentration 

(thousand//iil.)  in 

the  liquid  portion 

108  (±9) 



Volume  of  fluid  portion  (ml.) 

292  (±12) 



Composition  of  the  fluid  portion 

(mg./lOO  ml.) 


9  (±0-5) 




17  (±M) 



Citric  acid 

173  (±9) 



Lactic  acid 

21  (±3) 



Total  dry  weight 




Dry  wt.  of  non-dialysable 





Total  phosphorus 




Acid-soluble  phosphorus 
















Hexosamine  (after  acid 




(t^)  of  that  present  in  the 

epididymis,  and  the  concentrations  of 

fructose,  ergothioneine  and  citric  acid,  were 

20-9%  (tV),  20-9%  (il), 

and  20-8%  (|^),  respectively,  of  those 

present  in 

the  seminal 

vesicle  secretion.  In  semen  collected  from  the  boar  by  the  'split- 
ejaculate  method'  it  is  possible  to  distinguish  clearly  three  distinct 
fractions.  According  to   McKenzie  et  al.  (1938),  the  pre-sperm 

Chemical  and  Physical  Properties  of  Semen         37 

fraction  comprises  5-20%,  the  sperm-rich  fraction  30-50%,  and  the 
post-sperm  fraction  40-60%  of  the  total  ejaculate.  The  pre-sperm 
fraction  which  is  ejaculated  first,  consists  of  a  watery,  often  dis- 
coloured, more  or  less  sperm-free  secretion,  probably  of  urethral 


'   i  '■ 


Af  / 

V  '• 



N  '• 


l\     1  ^  ^\ 

W  \     i^B 


//    1                      ^' 




1    L    ' 


V  \    ' 


W\  ,. 

L       ,  V*  ,     \a    1 

12  3  4  5  6Min. 

Fig.  7.  Composition  of  boar  semen  fractions  collected  by  the  'split-ejaculate 
method*  at  half-a-minute  intervals. 

A,   sperm  number  xlO^/fraction  (• #);  B,  fructose  content, 

mg./fraction    (O O);    C,    ergothioneine    content,    m./fraction 

(• •). 

(Glover  &  Mann,  1954) 

38  The  Biochemistry  of  Semen 

origin.  Within  a  few  minutes,  that  is  followed  by  a  sperm-containing 
fraction  which  may  also  contain  some  gel-like  material.  As  a  rule 
however,  the  gel  forms  a  distinct  fraction  and  is  voided  shortly  after 
the  sperm  fraction.  Moreover,  these  three  fractions,  which  complete 
an  ejaculation  'wave',  may  be  succeeded  by  a  second  'wave',  also 
fractionated.  The  two  waves  together  may  last  up  to  30  min.,  and 
represent  in  fact,  two  successive  ejaculates.  By  the  estimation  of 
sperm  concentration  coupled  with  the  chemical  determination  of 
fructose,  citric  acid,  and  ergothioneine  in  the  various  fractions,  it  is 
possible  to  show  that  during  the  ejaculation  the  seminal  vesicle 
secretion  follows  immediately  upon  the  delivery  of  the  spermatozoa, 
and  is  found  mainly  in  the  sperm-containing  fraction  (Glover  and 
Mann,  1954;  Fig.  7).  Occasionally,  however,  the  ejaculation  of 
semen  is  incomplete  and  the  semen  does  not  include  the  later 
fractions.  When  this  happens,  for  example  in  the  stallion,  certain 
normal  constituents  such  as  fructose  or  citric  acid,  may  be  missing 
altogether.  Obviously,  such  incomplete  ejaculations  create  a  further 
complication  in  the  assessment  of  analytical  results  obtained  with 
semen,  at  any  rate  in  those  species  in  which  the  ejaculation  is  a 
fractionated  one.  But  even  in  the  bull  where  under  physiological 
conditions  ejaculation  appears  to  be  instantaneous,  the  'split- 
ejaculate  method'  applied  by  Lutwak-Mann  and  Rowson  (1953) 
with  the  aid  of  electric  stimulation,  demonstrated  the  occurrence  in 
electrically-induced  ejaculates,  of  at  least  two  distinct  fractions;  of 
these,  the  first  was  a  copious  sperm-free  fraction,  slightly  viscous, 
colourless,  and  of  urethral  origin,  in  which  there  was  no  fructose  or 
citric  acid  and  very  little  protein;  the  next  was  a  creamy  sperm- 
containing  fraction,  followed  by,  or  more  often  mixed  with,  a  yel- 
lowish-coloured post-sperm  fraction  which  represented  an  almost 
pure  secretion  of  the  seminal  vesicles,  with  a  characteristically  high 
content  of  fructose,  citric  acid,  and  5-nucleotidase. 

Criteria  for  the  rating  of  semen  quality 

General  medical  and  veterinary  experience  indicates  that  the  suc- 
cessful fertihzation  of  the  ovum  and  initiation  of  pregnancy,  while 
it  is  brought  about  primarily  by  the  spermatozoa  present  in  an 
ejaculate,  demands  nevertheless  the  attainment  of  certain  qualita- 
tive and  quantitative  standards  by  the  semen.  We  should  therefore, 

Chemical  and  Physical  Properties  of  Semen         39 

be  able  to  define  what  constitutes  normal  fertile  semen,  and  what 
criteria  if  any,  can  be  applied  in  the  appraisal  of  ejaculated  semen. 
It  may  be  stated  at  once  that  in  spite  of  the  wealth  of  information 
gained  by  past  and  present  students  of  semen,  there  is  as  yet  no  single 
seminal  characteristic  known,  which  alone  could  serve  as  the  means 
of  judging  'male  fertility'.  The  best  criterion  of  the  fertilizing  capacity 
of  spermatozoa  is  of  course,  the  actual  ability  to  fertilize  the  ovum. 
This  however,  cannot  be  regarded  as  a  laboratory  test,  until  the 
in  vitro  fertilization  of  the  ovum  has  actually  been  accomplished 
and  the  quantitative  aspects  of  the  process  developed. 

In  the  practice  of  artificial  insemination  of  cattle,  male  fertility 
continues  to  be  assessed  on  the  basis  of  the  'conception  rate'.  At 
the  artificial  insemination  centres,  inseminated  cows  which  have 
not  been  'returned'  by  the  farmers  for  re-insemination  within  three 
months  or  so,  are  presumed  to  have  conceived,  and  the  proportion 
of  presumed  pregnancies,  expressed  as  the  percentage  of  the  total 
of  the  first  inseminations,  is  referred  to  as  'conception  rate'.  Nearly 
one-third  of  the  cow  population  of  England  is  now  bred  by  artificial 
insemination,  and  among  these  the  conception  rate  averages  at  least 
60%.  There  is  considerable  evidence,  however,  that  a  substantial 
proportion  of  cows  'returned'  for  re-insemination,  may  have  also 
conceived  but  that  pregnancy  terminated  at  an  early  stage  through 
faulty  ovum  implantation  or  embryonic  death. 

Apart  from  the  test  based  upon  accomplished  fertilization,  the 
means  available  at  present  for  the  evaluation  of  semen  quality 
include  the  histological  and  the  physico-chemical  methods. 

Histological  examination  of  semen  involves  procedures  such  as 
the  determination  of  sperm  concentration  or  'density'  (number  of 
spermatozoa  per  \fj\.  or  1  ml.  of  semen)  with  a  cytometer  (Walton, 
1927;  Weisman,  1942);  differential  count  of  abnormal  forms  of 
spermatozoa  (Lagerlof,  1934;  Harvey  and  Jackson,  1945;  Lane 
Roberts  et  al.,  1948;  Williams,  1950);  bacteriological  examination 
(Gunsalus,  Salisbury  and  Willett,  1941;  Kelly,  1947;  Foote  and 
Salisbury,  1948;  Almquist,  Prince  and  Reid,  1949;  Wu,  Elliker  and 
McKenzie,  1952-3);  determination  of  the  incidence  of  dead  sperma- 
tozoa by  means  of  'live-dead  staining'  methods  (Lasley,  Easley  and 
McKenzie,  1942;  Lasley  and  Bogart,  1943;  Madden,  Herman  and 
Berousek,  1947;  Crooke  and   Mandl,  1949;  Blom,  1950;  Mayer, 

40  The  Biochemistry  of  Semen 

Squiers,  Bogart  and  Oloufa,  1951;  Ortavant,  Dupont,  Pauthe  and 
Roussel,  1952;  Campbell,  Hancock  and  Rothschild,  1953);  micro- 
scopic assessment  of  the  degree  of  motility,  either  directly  in  semen 
(Harvey  and  Jackson,  1945;  McLeod,  \9A6a;  Emmens,  1947;  Farris, 
1950)  or  by  the  'cervical  mucus  penetration  test',  in  which  a  drop  of 
semen  is  placed  on  the  microscope  slide  next  to  cervical  mucus  and 
the  passage  of  spermatozoa  through  the  mucus  is  followed  by  micro- 
scopic observation  (Barton  and  Wiesner,  1946;  Harvey  and  Jackson, 
1948).  The  determination  of  the  concentration  of  motile  spermatozoa 
in  a  semen  sample  is  generally  held  to  be  the  criterion  most  clearly 
correlated  with  the  actual  fertility  rate;  but  even  this  relationship 
occasionally  fails  to  give  a  true  picture,  and  motile  spermatozoa 
are  by  no  means  always  fertile. 

Physico-chemical  methods  of  semen  analysis  depend  on  the  deter- 
mination of  a  wide  range  of  physical,  chemical  and  metabolic 
characteristics  of  semen,  related  to  the  physiological  function  of  the 
sperm  and  the  seminal  plasma.  Here  belong  methods  for  the 
measurement  of  certain  optical  properties  of  semen  such  as  the 
light-scattering  and  light-absorption  power,  electrical  conductivity 
and  impedance  changes;  specific  gravity,  osmotic  pressure;  hydrogen 
ion  concentration,  buffering  capacity;  occurrence  of  semen-specific 
metabolites  such  as  fructose,  citric  acid,  ergothioneine,  and  inositol, 
and  various  enzymes  such  as  hyaluronidase  and  certain  phospha- 
tases; and  finally,  the  rate  of  anaerobic  and  aerobic  metabolic 
processes  in  semen  expressed  in  terms  of  fructolysis  index,  respira- 
tion rate  or  methylene-blue  reduction  time. 

Optical  and  electrical  properties  of  semen 

An  optical  property  closely  related  to  sperm  concentration  is  the 
turbidity  of  semen.  In  the  past,  determinations  of  sperm  concen- 
tration were  based  on  microscopic  sperm-counts  but  more  recently 
these  have  been  partly  replaced  by  turbidimetric  measurements, 
which  can  be  carried  out  quickly  with  suitably  diluted  samples  either 
in  a  visual  comparator  by  direct  comparison  with  opacity  standards, 
or  in  a  photoelectric  absorptiometer,  to  provide  values  for  the  rela- 
tive light  transmission  of  semen  (Burbank,  1935;  Comstock  and 
Green,  1939;  Henle  and  Zittle,  1942;  Salisbury  et  al.  1943;  Roth- 
schild,  19506).  The  turbidimetric  methods,  however,  it  must  be 

Chemical  and  Physical  Properties  of  Semen         41 

remembered,  rest  on  the  assumption  that  although  light  is  absorbed 
both  by  sperm  and  seminal  plasma,  the  scattering  of  light  is  due 
exclusively  to  the  former,  so  that  only  light-scattering  as  such,  is 
related  directly  to  sperm  density.  In  practice,  the  light-scattering  and 
light-absorption  due  to  the  spermatozoa  predominates  so  much 
over  the  light-absorption  of  the  seminal  plasma  that  under  properly 
controlled  conditions  the  error  due  to  the  presence  of  the  latter  can 
be  neglected.  This  applies  certainly  to  the  semen  of  sea-urchin,  bull 
and  ram,  but  need  not  necessarily  be  true  of  other  species,  in  which 
the  seminal  plasma  itself  shows  a  considerable  degree  of  opaqueness. 
Furthermore,  the  spermatozoa  present  in  a  given  ejaculate  may 
differ  in  their  light-reflecting  capacity,  apparently  in  proportion  to 
the  degree  of  sperm  'ripeness'  and  thereby,  of  fertility  (Lindahl 
et  al,  1952).  The  light-reflecting  capacity  of  the  sperm  cell  may 
also  be  related  to  another  optical  change  associated  with  sperm 
ripening,  namely  the  increase  in  'luminosity'  of  the  sperm  surface 
in  dark-field  illumination. 

It  has  been  claimed  that  the  spermatozoon  possesses  at  the  head 
and  tail  small,  but  directly  opposite,  electrical  charges.  However, 
all  that  can  be  claimed  with  certainty  is  that  an  electric  charge  is 
associated  with  the  sperm  cell  but  that  its  sign  and  magnitude  depend 
largely  on  the  concentration  of  the  various  positively  and  negatively 
charged  ions  in  the  surrounding  medium.  The  following  values  for 
electro-conductivity  in  semen  at  25°,  expressed  in  reciprocal 
ohmsxlO"*,  were  given  by  Bernstein  and  Shergin  (1936):  bull 
89-5-116-3,  ram  48-5-80-5,  stallion  111-3-129-5,  boar  123-3-134-6, 
rabbit  85-5-101 -4;  and  by  Zagami  (1939):  man  88-107  (at  20°),  and 
dog  129-138. 

Much  scientific  interest  and  practical  importance  in  the  rating  of 
semen  quality  attaches  to  the  characteristic  periodic  changes  in 
electrical  impedance  which  occur  in  semen  samples  with  high  sperm 
density  and  motility,  and  which  have  been  shown  by  Rothschild 
(1948^,  1949,  1950a)  to  be  associated  closely  with  the  so-called 
'wave  motion'  of  the  spermatozoa.  When  a  drop  of  ram  or  bull 
semen  of  high  density  is  placed  on  a  microscope  slide  and  examined 
at  37°  under  low  magnification,  a  characteristic  phenomenon  can 
be  observed  in  the  form  of  slow,  periodically  appearing  bands  of 
high  opacity  or  'waves'.  Measurements  by  means  of  the  impedance 

42  The  Biochemistry  of  Semen 

bridge  make  it  possible  to  assess  this  characteristic  sperm  movement 
in  a  more  objective  and  quantitative  manner  than  by  visual  esti- 
mates; but  they  can  only  be  made  with  semen  samples  which  are 
sufficiently  dense  to  show  the  'wave  motion'.  Assays  carried  out 
with  the  electrical  method  in  the  semen  of  bulls  at  several  British 
Artificial  Insemination  Centres  revealed  an  interesting  correlation 
of  impedance  change  frequency  with  the  conception  rate  (Bishop 
et  ai,  1954). 

Distinct  from  the  wave-motion  or  locomotion  'en  masse'  (Walton, 
1952)  is  the  movement  of  individual  spermatozoa  which  in  highly 
motile  semen  takes  the  form  of  so-called  'forward'  or  'progressive' 
motility,  but  which  in  poor  specimens  is  confined  to  side-to-side 
'oscillatory'  movements  (Plate  IV).  Recently,  there  have  been  several 
attempts  to  replace  the  subjective  and  semi-quantitative  microscope 
appraisals  of  motility  by  more  clearly  defined  methods,  of  which 
those  by  Bosselaar  and  Spronk  (1952)  and  Rothschild  (1953a,  b) 
deserve  special  mention. 

Viscosity,  specific  gravity,  osmotic  pressure,  and  ionic  equilibrium 

The  viscosity  of  whole  semen  depends  largely  upon  the  concen- 
tration of  spermatozoa.  Thus  for  example,  the  viscosity  of  bull 
semen  (relative  to  that  of  pure  water  which  is  taken  as  unity)  can 
vary  from  1-76  in  a  specimen  containing  80,000  sperm/ywl.  to  10-52 
in  a  sample  with  2,260,000  sperm //^l.  (Szumowski,  1948).  Seminal 
plasma  itself,  seldom  exceeds  in  bulls  a  relative  viscosity  value  of  2 
but  higher  values  have  been  recorded  in  other  species,  especially 
those  which  exhibit  the  phenomenon  of  gelation. 

The  average  specific  gravity  of  whole  semen  is  1028  in  man, 
1-011  in  dog,  and  1-035  in  bull,  with  fluctuations  due  in  the  first 
place  to  the  variable  ratio  between  sperm  and  seminal  plasma.  The 
latter  is  so  much  lighter  than  the  spermatozoa  that  in  practice  the 
specific  gravity  of  semen  is  often  found  to  be  directly  proportional 
to  sperm  concentration.  In  bull  semen,  low  specific  gravity  is  usually 
associated  with  low  sperm  concentration  and  poor  'quality',  whereas 
high  values  accompany  good  density  and  good  'quality'  (Anderson, 
1946(7).  This  is  not  surprising  in  view  of  the  fact  that  the  specific 
gravity  of  bull  seminal  plasma  is  not  greater  than  that  of  blood 
plasma,  whereas  the  average  specific  gravity  of  bull  spermatozoa 



Cinematograph  of  a  bull  spermatozoon  (in  semen  diluted  1  :  450)  moving 
forward  at  a  speed  of  about  0-15  mm. /sec.  Photographic  plate 
exposed  for  1  sec,  using  dark  ground  illumination.  Mag. x  673.  Only 
the  projection  of  the  movement  of  the  sperm-head  is  seen,  the  tail 
leaving  no  track.  As  the  sperm-head  is  shaped  like  an  elliptical  disc, 
intense  light  scattering  occurs  only  when  the  thin  edge  of  the  head 
is  visible;  during  1  sec.  exposure  ten  images  of  the  head  were  recorded 
which  means  that  it  rotated  or  oscillated  backwards  and  forwards 
ten  times. 

(By  courtesy  of  Lord  Rothschild) 

Chemical  and  Physical  Properties  of  Semen         43 

(1-28)  considerably  exceeds  that  of  the  erythrocytes  (11)  and  other 
cells  of  the  animal  body.  As  already  mentioned  (p.  8),  Lindahl 
and  Kihlstrom  (1952)  believe  that  the  wide  range  of  variations  in  the 
specific  gravity  of  ejaculated  bull  spermatozoa  (1-240-1 -334)  is  due 
to  the  variable  proportion  of  'ripe'  (heavier)  and  'unripe'  (lighter) 
sperm  cells  in  semen.  However,  whereas  the  specific  gravity  of  the 
sperm  cell  is  due  to  the  highly  condensed  nuclear  and  protoplasmic 
protein  constituents,  the  specific  gravity  of  the  seminal  plasma  is  the 
direct  outcome  of  the  actual  osmotic  pressure  exerted  by  electrolytes 
and  is  thus  related  to  the  depression  of  the  freezing  point. 

Determinations  of  the  osmotic  pressure  in  terms  of  freezing  point 
depression  have  been  carried  out  in  the  semen  of  several  species  and 
the  following  data  (in  centigrade)  are  available:  man  0-55-0-58,  bull 
0-54-0-73,  ram  0-55-0-70,  stallion  0-58-0-62,  jackass  0-56-0-62,  boar 
0-59-0-63,  dog  0-58-0-60,  rabbit  0-55-0-59  (Slovtzov,  1916;  Roem- 
mele,  1927;  Milovanov,  1934;  Bernstein  and  Shergin,  1936;  Zagami, 
1940;  Salisbury,  Knodt  and  Bratton,  1948;  Nishikawa  and  Waide, 
1951).  It  would  seem  that  generally,  more  reliance  should  be  placed 
on  results  obtained  with  seminal  plasma  than  with  whole  semen. 
More  recently,  Rothschild  and  Barnes  (1954)  carried  out  freezing 
point  determinations  on  forty  samples  of  seminal  plasma  from  ten 
bulls  of  different  breeds,  and  obtained  a  mean  value  of  0-53  with  a 
standard  error  of  0-005. 

The  electrolytes  in  the  seminal  plasma  are  those  made  available 
by  the  secretions  of  the  male  accessory  organs.  Additional  informa- 
tion on  the  content  of  the  inorganic  constituents  in  semen  is  sum- 
marized in  Table  4,  but  again,  owing  to  the  variable  composition  of 
semen,  the  chemical  data  relating  to  whole  semen  must  be  taken  with 
due  reservations. 

The  interrelations  between  the  various  ions  in  semen  differ  from 
those  existing  in  blood  in  several  respects,  but  most  perhaps  because 
of  the  presence  of  a  much  higher  concentration  of  extracellular 
potassium  and  a  correspondingly  lower  content  of  sodium.  Miescher 
(1897)  who  was  the  first  to  examine  systematically  the  chemical 
composition  of  salmon  semen,  found  in  the  ash  prepared  from  the 
seminal  plasma  (parts/100):  51-0  NaCl,  8-2  KCl,  140  K2SO4,  and 
26-8  Na.COa.  In  the  trout,  Schlenk  (1933)  recorded  a  value  of  80  mg. 
K/lOO  ml.  semen.  In  the  sea-urchin  {Echinus  esculent  us)  Rothschild 


The  Biochemistry  of  Semen 

(1948c)  found  155  mg.  K/lOO  ml.  seminal  plasma.  In  the  higher 
mammals,  the  content  of  potassium  in  semen  may  reach  400  mg./ 
100  ml.  (Table  4);  it  is  derived  mainly  from  the  seminal  plasma  where 
it  is  found  at  least  partly,  in  association  with  citric  acid  (see  p.  188). 

Hydrogen  ion  concentration  and  buffering  capacity 

The  reaction  of  freshly  ejaculated  semen  is  not  far  from  neutral 
(Table  8).  On  standing,  the  semen  may  become  alkaline  at  first, 
unless  precautions  are  taken  to  prevent  the  loss  of  carbon  dioxide, 

Table  8.  Hydrogen  ion  concentration  in  semen 




7-3  -7-9 


6-4  -7-8 




6-3  -7-8 




6-2  -6-4 






6-8  -7-5 


5-9  -7-3 


6-2  -7-8 

Arbacia  pimctidata 

7-6  -7-9 

Echinus  escidentus 



McKenzie,  Miller  and  Bauguess  (1938) 

Anderson  (1942);  Hatziolos  (1937) 

Laing  (1945) 

Zagami  (1939) 

Lambert  and  McKenzie  (1940) 

Zagami  (1939) 

Starkov  (1934) 

Zagami  (1939) 

Huggins,  Scott  and  Heinen  (1942) 

Zagami  (1939) 

Lambert  and  McKenzie  (1940) 

McKenzie  and  Berliner  (1937) 

Nishikawa  and  Waide  (1951) 

Hayashi  (1945) 

Rothschild  (1948c) 

but  later  this  change  is  followed,  at  least  in  those  specimens  which 
contain  fructose  and  a  high  concentration  of  spermatozoa,  by  a 
rapid  decrease  of  pH,  owing  to  fructolysis  and  accumulation  of 
lactic  acid.  Excessive  initial  alkalinity  of  semen  in  some  species, 
notably  in  bulls  and  rams,  often  accompanies  low  fertility,  the 
alkaline  reaction  being  associated  with  absence  or  low  concen- 
tration of  sperm  and  with  a  correspondingly  higher  proportion  of 
seminal  plasma.  A  significant  negative  correlation  between  the  pH 
value  and  sperm  density  and  motility  has  been  noted  frequently 
in  bulls  and  rams  (cf.  Anderson,  1945);  in  the  latter,  according  to 
McKenzie  and  Berliner  (1937),  normal  semen  is  slightly  acid  or  at 

Chemical  and  Physical  Properties  of  Semen         45 

any  rate  never  more  alkaline  than  pH  7-3,  whereas  in  sterile  rams  the 
pH  value  may  reach  8-6.  However,  the  rate  at  which  acidity  increases 
after  ejaculation  is  much  more  significant  for  the  assessment  of 
semen  than  the  initial  pH  value,  because  it  is  related  directly  to  the 
actual  glycolytic  activity  of  the  spermatozoa,  and  indirectly  to 
sperm  density  and  motility.  In  bull  and  ram  semen  this  correlation 
is  said  to  be  so  close  that  the  decrease  in  pH  on  incubation  can 
serve  as  an  additional  indicator  of  semen  quality  (cf.  Anderson, 
1945;  Laing,  1945;  Reid,  Ward  and  Salsbury,  1948Z)).  As  a  matter 
of  fact,  however,  the  significance  of  this  method  is  limited  because 
as  the  pH  value  falls,  the  semen  becomes  too  acid  for  spermatozoa 
to  maintain  their  motility  and  metabolism. 

Some  of  the  effects  which  are  due  to  variations  in  hydrogen  ion 
concentration  in  semen  will  be  discussed  in  the  next  chapter;  here 
we  shall  concern  ourselves  with  the  buffering  capacity  of  semen 
which  has  been  the  subject  of  some  interesting  work,  especially  in 
bull  and  human  semen  (Shergin,  1935;  Smith  and  Asdell,  1940; 
Easley,  Mayer  and  Bogart,  1942;  Sheldovsky,  Belcher  and  Leven- 
stein,  1942;  Willett  and  Salisbury,  1942;  Anderson,  1946«).  On  the 
whole,  bull  semen  is  more  highly  buffered  on  the  acid  than  on  the 
alkaline  side,  and  its  normal  buffering  capacity  depends  mainly 
upon  citrate  and  bicarbonate,  but  not  phosphate.  Anderson  used 
107  specimens  of  seminal  plasma  from  normal  bulls  and  measured 
their  buffering  power  by  adding  005  ml.  OIn-HCI  to  01 5  ml. 
plasma,  and  determining  the  pH  with  the  help  of  a  glass  electrode, 
before  and  after  acid  addition;  the  decrease  in  pH  value  obtained 
in  this  way  was  l-84±0038.  He  also  investigated  the  relationship 
between  buffering  capacity  and  the  period  for  which  sperm  motility 
of  70%  and  over,  was  maintained;  semen  specimens  which  kept  up 
this  motility  for  24  hr.  and  upward,  all  had  a  good  and  fairly  uniform 
buffering  capacity  (decrease  in  pH=l-74),  but  those  with  a  smaller 
degree  of  motility  at  24  hr.  had  a  poorer  buffering  capacity  (decrease 
in  pH=200). 

Metabolism  of  semen,  and  its  relation  to  sperm  density  and  motility 

The  two  chief  metabolic  processes  of  semen,  namely  fructolysis 

and  respiration,  are  both  a  direct  outcome  of  the  metabolic  activity 

of  the  sperm  cells;  their  rate  is  determined  largely  by  the  number 

46  The  Biochemistry  of  Semen 

of  spermatozoa  in  the  semen  and  the  degree  of  sperm  motility 
(Mann,  1949).  The  more  chemical  aspects  of  semen  metabolism 
will  be  discussed  fully  in  conjunction  with  specific  groups  of  sub- 
stances metabolized  by  spermatozoa,  such  as  sugars,  lipids  and 
amino  acids.  Here,  only  the  general  outline  of  sperm  metabolism 
will  be  given,  in  so  far  as  it  helps  to  bring  out  the  relationship 
between  the  metabolic  processes  and  other  characteristics  of  semen, 
in  particular,  sperm  concentration  and  motility. 


In  the  absence  of  oxygen,  for  example  under  the  conditions  of 
semen  storage  for  artificial  insemination,  the  spermatozoa  rely  on 
carbohydrate  metabolism  as  the  chief  source  of  energy.  Even  before 
the  identity  of  the  seminal  sugar  was  revealed,  the  rate  of  lactic  acid 
production  or  'glycolysis'  was  used  as  a  method  for  semen  appraisal 
(Comstock,  1939;  Webster,  1939;  MacLeod,  1941a,  b,  1943Z);  Moore 
and  Mayer,  1941;  Comstock,  Green,  Winters  and  Nordskog,  1943; 
Laing,  1945;  Salisbury,  1946;  Westgren,  1946.)  However,  the  dis- 
covery of  fructose  and  the  work  on  fructolysis  made  available  a 
chemical  approach  to  several  practical  problems  of  male  fertility 
(Mann,  19466,  1948«,  b,  1949;  Mann,  Davies  and  Humphrey,  1949; 
Mann  and  Lutwak-Mann,  \95\a,  b;  Mann  and  Parsons,  1950; 
Mann  and  Walton,  1953). 

The  anaerobic  incubation  of  freshly  ejaculated  semen  is  accom- 
panied by  a  progressive  decline  in  the  content  of  fructose  with  a 
simultaneous  accumulation  of  lactic  acid;  in  the  presence  of  suitable 
buffer,  the  process  of  fructolysis  in  semen  with  good  sperm  motility 
can  be  shown  to  progress  almost  linearly  until  practically  all  of  the 
sugar  is  used  up  (Fig.  8).  On  this  basis  a  photometric  method  has 
been  worked  out  for  the  measurement  of  sperm  fructolysis,  and  the 
'index  of  fructolysis'  has  been  defined  as  the  amount  of  fructose  (in 
mg.)  utilized  by  10^  spermatozoa  in  1  hr.  at  37°  (Mann,  1948^,  b). 
In  normal  bull  semen,  the  index  of  fructolysis  is  about  1  -4-2  but  it 
varies,  and  is  significantly  correlated  with  both  the  concentration 
and  the  motility  of  spermatozoa.  Fructose  is  not  utilized  by  either 
azoospermic  semen,  i.e.  ejaculates  devoid  of  sperm,  or  by  necro- 
spermic  semen,  containing  immotile  spermatozoa.  The  existence 
of  a  positive  correlation  between  the  rate  of  fructolysis  and  the 

Chemical  and  Physical  Properties  of  Semen         47 

concentration  of  motile  spermatozoa  has  been  amply  confirmed  by 
studies  on  both  bull  and  human  semen  (Anderson,  1946b;  Eichen- 
berger  and  Goossens,  1950;  Bishop  et  al.,  1954)  but  the  existence  of 
a  similar  correlation  between  the  rate  of  fructolysis  and  fertility 

100  - 

Control  with  buffered  semen 
inactivated  by  heating 

30         60         90  120       150         180 

Minutes  of  incubation  at  37°  C 

210      240 

Fig.  8.  Fructolysis  in  bull  semen  (920,000  sperm/fil.)  incubated  at  37°; 
the  disappearance  of  fructose  was  measured  in  (i)  bull  semen  which 
has  been  diluted  with  half  a  volume  0-25m  phosphate  buffer  pH  7-4, 
(ii)  unbuffered  semen,  and  (iii)  buffered  semen  inactivated  by  heating. 

(Mann,  1948a) 

is  still  a  matter  of  dispute;  it  has  been  claimed  for  bull  semen  by 
some  authors  (Gassner,  Hill  and  Sulzberger,  1952)  but  was  denied 
by  others  (Bishop  et  al.,  1954).  In  both  cases,  however,  the  material 
used  for  the  survey  consisted  mainly  of  bulls  kept  for  breeding 
purposes  at  Artificial  Insemination  Centres.  More  insight  into  the 
relationship  between  fructolysis  and  fertility  could  probably  be 
gained  by  studies  on  semen  from  bulls  of  subnormal  fertility. 

48  The  Biochemistry  of  Semen 

A  lowered  rate  of  fructolysis  has  been  observed  in  certain  cases 
of  human  subfertiHty  (Davis  and  McCune,  1950;  Birnberg,  Sherber 
and  Kurzrok,  1952).  However,  in  human  semen  with  its  physio- 
logically low  sperm  density  but  high  fructose  content,  it  is  actually 
more  convenient  to  assess  the  rate  of  fructose  utilization  by  measur- 
ing the  formation  of  lactic  acid  (chemically  or  manometrically) 
rather  than  the  disappearance  of  sugar.  In  any  case,  if  one  were  to 
measure  fructolysis  in  human  semen  by  the  disappearance  of  sugar, 
it  would  be  essential  to  use  a  photometric  method  specific  for 
fructose,  and  not  base  the  results  merely  upon  the  changes  in  'reduc- 
ing value',  because  human  semen  contains  a  fair  amount  of  reducing 
substances  other  than  carbohydrate,  which  represent  a  substantial 
proportion  of  the  total  'reducing  value'  towards  reagents  such  as 
cupric  hydroxide,  ferricyanide  etc.  Moreover,  the  content  of  these 
reducing  substances  often  increases  during  the  incubation  of  semen, 
thus  rendering  unreliable,  not  to  say  senseless,  determinations  of 
fructose  based  upon  reduction  measurements. 

Methylene-blue  reduction  test 

A  method  often  used  in  the  evaluation  of  semen  quality  is  the 
'methylene-blue  reduction  test'  which  is  the  outcome  of  dehydro- 
genase activity  of  the  semen  and  depends  on  the  determination  of 
the  time  which  it  takes  a  semen  sample  to  decolorize  a  certain 
amount  of  methylene  blue,  under  standard  conditions  of  incuba- 
tion in  vitro  (S^rensen,  1942;  Beck  and  Salisbury,  1943;  VanDemark, 
Mercier  and  Salisbury,  1945;  Boenner,  1947).  In  S0rensen's  original 
method  the  incubation  was  carried  out  in  a  Thunberg  tube,  but  in 
Russia  (Milovanov  and  Sokolovskaya,  1947)  and  in  France  (Brochart, 
1948Z)),  the  test  was  later  performed  by  introducing  a  drop  of  semen 
mixed  with  methylene  blue  into  a  capillary  tube  and  by  observing 
the  decolorization  in  the  central  portion  of  the  column;  in  good- 
quality  bull  semen,  with  high  density  and  motility,  decolorization 
usually  takes  place  within  less  than  10  min.  at  20°;  if  it  extends 
beyond  30  min.,  it  signifies  poor  semen  quality.  However,  though 
useful,  the  test  is  of  limited  scope,  not  least  because  as  long  ago  as 
1941,  Lardy  and  Phillips  showed  that  the  reduction  of  methylene 
blue  by  sperm  suspensions  may  be  markedly  delayed  by  a  variety 
of  substances  including  glucose,  lactate  and  citrate. 

Chemical  and  Physical  Properties  of  Semen         49 


In  the  presence  of  oxygen,  semen  shows  a  considerable  respiratory 
activity  which  is  correlated  both  with  concentration  and  motility 
of  spermatozoa.  It  is  usual  to  express  sperm  respiration  in  terms  of 
Z02,  a  coefficient  introduced  by  Redenz  (1933)  to  denote  ^1.  O2  taken 
up  by  10^  sperm  cells  during  1  hr.  at  37°;  Z02  values  reported  by 
Lardy  and  Phillips  (1943a)  for  bull,  cock,  rabbit  and  ram  semen,  are 
21,  7,  11  and  22,  respectively.  The  use  of  Z02  is  more  convenient 
than  Q02  since  the  latter  involves  centrifugation  of  semen  and 
washing  of  sperm  with  an  unphysiological  fluid  such  as  distilled 
water;  the  average  Q02  of  ram  sperm  based  on  the  dry  weight  of 
washed  sperm  is  about  8.  Further  references  to  the  subject  of  sperm 
respiration  measurements,  classified  according  to  species  are  listed 
in  Table  9. 

Chang  and  Walton  (1940)  found  a  close  relationship  between 
motility  and  respiratory  activity  in  ram  sperm.  Walton  (1938)  sug- 
gested measurements  of  oxygen  uptake  in  bull  semen  as  a  supporting 
method  for  the  assessment  of  semen  quality.  Walton  and  Edwards 
(1938)  compared  the  breeding  records  of  thirteen  bulls,  taking  as  a 
measure  of  their  fertility  the  number  of  matings  required  to  produce 
pregnancy  in  cows;  when  they  analysed  ten  different  samples  of 
semen  from  each  of  these  bulls,  they  found  that  there  is  a  close  cor- 
relation between  the  respiratory  activity  of  semen  and  fertility 
assessed  on  the  natural  service  records.  But  Ghosh,  Casida  and 
Lardy  (1949),  and  Bishop  et  at.,  (1954)  failed  to  establish  a  corre- 
lation between  the  respiratory  activity  of  bull  semen  and  fertility, 
as  assessed  on  the  basis  of  artificial  insemination  records. 

Further  study  will  probably  clear  up  these  uncertainties  but  in 
general,  when  we  consider  the  significance  of  sperm  respiration 
measurements,  there  are  certain  points  which  must  be  taken  into 
account.  Unlike  fructolysis,  the  respiratory  activity  of  semen  is  not 
entirely  exogenous  since  it  involves,  in  addition  to  the  oxidative 
removal  of  products  of  fructolysis  (chiefly  lactic  acid),  the  endo- 
genous respiration,  i.e.  oxidation  of  some  intracellular  reserve 
material,  most  probably  a  lipid.  Moreover,  it  is  possible  to  create, 
experimentally  at  any  rate,  conditions  under  which  sperm  respira- 
tion can  be  dissociated  from  motility.  For  example,  in  the  presence 

50  The  Biochemistry  of  Semen 

Table  9.  List  of  some  references  to  work  on  sperm  respiration 


Winchester  and  McKenzie,  1941. 


Redenz,  1933;  Windstosser,  1935;  Henle  and  Zittle,  1941,  1942;  Lardy 
and  Phillips,  1943a,  b,  1944;  Lardy,  Hansen  and  Phillips,  1945;  Tosic 
and  Walton,  1950;  Schultze  and  Mahler,  1952;  Bishop  and  Salisbury, 
1954;  Melrose  and  Terner,  1953;  Bishop,  Campbell,  Hancock  and 
Walton,  1954. 


Winberg,  1939;  Lardy  and  Phillips,  1943a;  Kosin,  1944. 


Ivanov,  1931;  Bishop,  1942. 


Bishop,  1942. 


McLeod,  1939,  1943a,  b;  Shettles,  1940. 


Lardy  and  Phillips,  1943a;  White,  1953. 


Ivanov,  1936;  Comstock,  1939;  Chang  and  Walton,  1940;  Comstock, 
Green,  Winters  and  Nordskog,  1943;  Lardy,  Winchester  and  Phillips, 
1945;  Mann,  1945a;  Mann  and  Lutwak-Mann,  1948;  White,  1953. 
OYSTER,  Saxostrea  commercialis 
Humphrey,  1950. 


Warburg,  1915;  Gray,  1928,  1931;  Barron  et  ai,  1941,  1948,  1949; 
Hayashi,  1946;  Rothschild,  1948a,  c,  d,  1950^/,  I95\b;  Spikes,  1949. 

of  a  suitable  concentration  of  fluoride,  one  can  abolish  both  motil- 
ity and  fructolysis  in  ram  spermatozoa  without  greatly  suppressing 
the  respiration  (Mann  and  Lutwak-Mann,  1948)  (Fig.  9).  Another 
example  is  provided  by  the  response  of  ram  sperm  to  succinate. 
Thus,  whereas  the  oxygen  consumption  of  intact  ram  spermatozoa 
is  not  enhanced  markedly  by  the  addition  of  succinate,  sperm  cells 
treated  with  spermicidal  detergents  such  as  cetyltrimethylammonium 
bromide,  2-phenoxyethanol,  sodium  dodecylsulphate  and  similar 
surface-active  agents,  show  in  the  presence  of  succinate  a  high  rate 
of  oxygen  uptake  although  of  course,  the  motility  and  the  fructolysis 
have  been  completely  abolished  (Koefoed-Johnsen  and  Mann,  1954). 
The  effect  of  fluoride  on  fructolysis  is  due  chiefly  to  the  inhibition 
of  enolase;  the  addition  of  pyruvate  to  fluoride-treated  spermatozoa 

Chemical  and  Physical  Properties  of  Semen         5 1 

enables  the  lactic  acid  formation  to  continue  (Ivanov,  1943;  Lardy 
and  Phillips,  1943c;  Mann,  \9A5b\  Melrose  and  Terner,  1952,  1953). 
But  according  to  the  last-named  authors,  washed  bull  spermatozoa 

1                        I                      T- 



/       a    - 
/       o 
/            3 

/                     bb 

'                  E 



pe  - 












/    .^y 


/     ^y 


/             ^  y 


/          ^  / 



E       / 

1^     / 
r^    / 

f     ^-y 








Fig.  9.  Effect  offiiioride  on  the  respiration  and  aerobic  fructolysis  of  ram 
semen.  Each  manometer  flask  contained  0-5  ml.  whole  semen  diluted 
with  1  -5  ml.  Ringer-phosphate  solution.  Arrows  show  fructose  content. 

(Mann  &  Luwak-Mann,  1948) 

incubated  with  fluoride  and  pyruvate  under  strictly  anaerobic  con- 
ditions, are  unable  to  convert  pyruvic  acid  entirely  to  lactic  acid 
but  instead,  a  dismutation  takes  place  in  which  of  two  molecules 
pyruvic  acid,  only  one  is  reduced  to  lactic  acid,  and  the  other  is 
oxidized  to  carbon  dioxide  and  acetic  acid: 
2CH3  CO  COOH  +  H2O  — >  CH3  CHOHCOOH  +  CO^  +  CH3COOH 

52  The  Biochemistry  of  Semen 

Aerobically,  in  the  presence  of  both  fluoride  (0  02m)  and  pyruvate 
(001m),  bull  spermatozoa  consume  less  oxygen  than  without 
fluoride;  this  low  rate  of  oxygen  uptake  can  be  enhanced  by  the 
addition  of  2  :  4-dinitrophenol  (10~*m),  which  brings  about  a  more 
complete  oxidation  of  that  fraction  of  pyruvate  which  is  not  reduced 
to  lactic  acid.  Melrose  and  Terner  (1952,  1953)  claim  that  bull 
semen  can  be  graded  according  to  the  respiratory  response  of  washed 
spermatozoa  to  a  system  made  up  of  fluoride,  pyruvate  and  dini- 
trophenol,  and  that  in  highly  fertile  samples  the  oxygen  consumption 
is  low  in  the  presence  of  pyruvate  and  fluoride,  but  is  increased  two- 
fold or  more,  by  the  addition  of  dinitrophenol. 

Among  substances  which  can  provide  exogenous  material  for 
sperm  respiration  are  glycolysable  sugars  and  lower  fatty  acids  such 
as  lactic,  pyruvic  and  acetic  acid  (Fig.  10)  but  the  species  differences 
in  this  respect  are  very  marked.  Thus,  while  in  mammalian  sperma- 
tozoa for  example,  it  is  possible  to  prolong  the  respiratory  activity 
for  a  considerable  length  of  time  with  glucose,  fructose,  mannose, 
L(+)-lactate,  pyruvate,  propionate,  butyrate,  and  oxaloacetate 
(Lardy  and  Phillips,  1944,  1945;  Mann  and  Lutwak-Mann,  1948; 
Humphrey  and  Mann,  1949;  Tosic  and  Walton,  1950;  Melrose  and 
Terner,  1953),  the  respiration  of  oyster  spermatozoa  is  increased  by 
a-oxoglutarate  and  oxaloacetate,  decreased  by  acetate,  propionate 
and  butyrate,  and  remains  unaffected  by  lactate,  glucose,  fructose 
and  mannose  (Humphrey,  1950).  It  appears  also  that  the  mode  of 
action  of  several  organic  acids  on  the  respiration  of  mammalian 
spermatozoa  differs  fundamentally  from  the  influence  exerted  by  the 
same  substances  on  the  sperm  of  lower  animals.  This  is  illustrated 
best  by  the  example  of  the  peculiar  response  of  sea-urchin  sperm 
to  malonate  (003m),  which  was  reported  by  Barron  and  Goldinger 
(1941/))  to  increase  both  the  O2  uptake  and  the  aerobic  CO2  output 
of  sperm  by  as  much  as  200%.  Succinic  acid  is  another  example  of  a 
substance  which  was  found  to  be  highly  effective  in  the  respiration 
of  sea-urchin  sperm  (Barron  and  Goldinger,  \94\a;  Goldinger  and 
Barron,  1946)  but  has  little  effect  on  the  oxygen  uptake  of  intact 
mammalian  spermatozoa.  Another  characteristic  difference  in  be- 
haviour between  sea-urchin  and  mammalian  sperm  concerns  the 
effect  of  the  fatty  acids  on  the  initial  rate  of  sperm  respiration.  In 
the  case  of  sea-urchin  spermatozoa,  the  addition  of  a  fatty  acid  salt 

Chemical  and  Physical  Properties  of  Semen 








-•  Fructose 

o o  Glucose 

X X  Na-lactate 


210      240 

90  120        150 

Incubation  (min.) 
Fig.  10.  Effect  of  fructose,  glucose  and  lactate  on  the  respiration  of  washed 
ram  spermatozoa  (the  same  effect  was  obtained  also  with  acetate  and 
pyruvate);  oxygen  uptake  of  3  ml.  buffered  suspension  of  0-45x10^ 

(Mann  &  Lutwak-Mann,  1948) 

often  produces  a  prompt  and  marked  rise  in  the  initial  rate  of  O2 
uptake.  In  our  experience,  however,  the  addition  of  organic  acid 
salts  to  washed  suspensions  of  mammalian  spermatozoa,  does  not 
lead  necessarily  to  an  actual  increase  in  the  initial  O2  uptake  but 
instead,  these  substances  act  by  maintaining  and  prolonging  the 
initial  rate  of  respiration,  thereby  delaying  the  decline  in  Oo  con- 
sumption which  would  set  in  otherwise  (Mann,  1949).  Furthermore, 
by  adding  the  same  substances  to  respiring  suspensions  of  washed 
mammalian  sperm  at  a  stage  when  the  respiration  had  already  begun 
to  decline,  one  can  prevent  a  further  deterioration  in  the  rate  of  O2 


77?^  Influence  of  Extraneous  Factors, 
Hormones,  and  Environmental  Conditions 

Sperm  inhibitors  and  spermicidal  substances.  Cliemical  aspects  of  short- 
wave radiation.  Variations  in  hydrogen  ion  concentration  and  tonicity. 
Influence  of  heat  and  cold;  sperm  vitrification  and  'la  vie  latente'.  Role  of 
hormones.  Sperm-egg  interacting  substances  and  chemotaxis.  'Dilution 
effect'  and  chemical  changes  associated  with  senescence.  The  use  of  arti- 
ficial diluents  in  the  storage  of  semen. 

The  list  of  agents,  both  physical  and  chemical,  which  affect  sperma- 
tozoa, includes  among  others,  changes  in  temperature,  visible  light, 
short-wave  radiation,  atmospheric  pressure,  ionic  strength,  and  a 
host  of  pharmacologically  active  substances.  The  vast  literature  on 
the  subject  of  sperm  activation  and  inhibition  goes  back  as  far  as 
Leeuwenhoek's  observation  that  dilution  with  rain  water  deprives 
the  canine  'animalculi'  of  motion,  and  a  report  by  his  learned  friend 
Johan  Ham  of  Arnhem,  on  the  loss  of  sperm  motility  in  a  patient 
dosed  with  turpentine.  Among  Spallanzani's  contributions  in  this 
field  is  the  discovery  that  freezing  in  snow  does  not  necessarily  kill 
the  'spermatic  vermiculi'  but  reduces  them  to  a  state  of  'lethargy' 
from  which  they  recover  when  returned  to  higher  temperature.  The 
XlXth  century  abounds  in  studies  on  the  effect  of  changes  in  the 
medium  on  sperm  motility  and  survival.  Prevost  and  Dumas  (1824) 
extended  Spallanzani's  observations  on  the  lethal  effect  of  electric 
shock  and  certain  poisons;  Donne  (1837)  investigated  the  influence 
of  milk,  urine,  and  the  vaginal  and  cervical  secretions;  de  Quatre- 
fages  (1850,  1853)  described  in  great  detail  the  marked  toxicity  of 
copper,  lead  and  mercuric  salts.  Newport  (1853)  studied  the  nar- 
cotizing influence  of  chloroform  vapours  on  the  amphibian  sperma- 
tozoa, and  concluded  'that  the  spermatozoon  does  not  impregnate 
when  entirely  deprived  of  its  power  of  motion  by  narcotization  and 
disenabled  to  penetrate  into  the  envelopes  of  the  egg'.  Both  Newport 


The  Influence  of  Extraneous  Factors  55 

and  de  Quatrefages  were  fully  aware  of  the  fact  that  sodium  chloride 
and  various  other  sodium  and  potassium  salts,  are  able  to  stimulate 
or  inhibit  sperm  motility,  according  to  concentration  and  specific 
experimental  conditions.  Their  results  were  soon  confirmed  and 
extended  by  others,  including  Koelliker  (1856)  whose  paper  on 
'Physiologische  Studien  liber  die  Samenfllissigkeit'  still  remains  the 
most  comprehensive  survey  of  its  kind,  and  includes  observations 
on  spermatozoa  of  the  bull,  stallion,  dog,  rabbit,  pigeon,  frog  and 
fish.  Koelliker  pointed  out  that  spermatozoa  rendered  motionless  by 
dilution  with  water  can  be  revived  by  prompt  addition  of  salts  or 
concentrated  solutions  of  certain  organic  substances  such  as  sucrose, 
glucose,  lactose,  glycerol,  urea  and  various  proteins.  He  investi- 
gated in  some  detail  the  activating  influence  of  blood  serum,  male 
accessory  gland  secretions,  and  of  a  variety  of  inorganic  and  organic 
substances  on  sperm  motility.  It  was  he  who  found  that  cyanide  is 
not  an  inhibitor  of  sperm  motility  and  established  that  acids  are, 
on  the  whole,  more  harmful  to  the  sperm  than  alkalies. 

Furthermore,  Koelliker  noticed  that  if  a  drop  of  a  fairly  concen- 
trated solution  of  potassium  hydroxide  is  mixed  with  a  drop  of 
semen  on  a  microscopic  slide,  there  is  usually  a  sudden  outburst  of 
activity  before  the  spermatozoa  are  rendered  motionless.  Such  a 
period  of  short-lived  stimulation  which  precedes  the  terminal  loss 
of  activity,  is  rather  characteristic  of  various  sperm-paralysing 
agents  including  distilled  water.  Schlenk  (1933)  aptly  named  the 
phenomenon  'Todeszuckung'.  Not  all  investigators,  however,  seem 
to  have  realized  the  fundamental  difference  between  short  and  pro- 
longed activation  phenomena,  and  many  of  them  tended  to  confuse 
a  transient  increase  in  initial  motility  with  the  state  of  activity  essen- 
tial for  the  maintenance  of  continuous  motility  and  for  sperm  sur- 
vival. Only  too  often  substances  have  been  pronounced  as  beneficial 
to  spermatozoa  merely  because  they  were  observed  to  stimulate 
motility  and  metabolism,  no  heed  being  paid  to  the  fact  that  this 
very  stimulation  may  have  shortened,  rather  than  prolonged,  the 
life  of  spermatozoa.  Similarly,  many  a  substance  has  been  declared 
detrimental  to  spermatozoa  solely  because  it  appeared  to  reduce  the 
speed  of  movement  and  metabolic  rate.  However,  quite  often  the 
lowering  of  activity  tends  to  prolong  the  life  of  spermatozoa,  and 
favours,  rather  than  hinders,  their  survival. 

56  The  Biochemistry  of  Semen 

Sperm  inhibitors  and  spermicidal  substances 

So  far,  detailed  studies  on  the  mechanism  which  underlies  the 
action  of  sperm  inhibitors,  have  been  relatively  limited  in  scope  and 
concerned  largely  with  chemical  compounds  which  affect  respiration 
and  glycolysis,  among  them  cyanide,  azide,  dinitrophenol,  and 
fluoride,  or  which  combine  with  sulphydryl  groups,  e.g.  iodoacetate, 
iodoacetamide,  <?-iodosobenzoate,  and  /7-chloromercuribenzoate. 
Even  these  studies,  however,  have  clearly  indicated  the  existence  of 
remarkable  species  differences  in  sperm  behaviour.  Thus  for  instance, 
iodoacetate  which  is  one  of  the  strongest  inhibitors  of  sperm  activity 
in  higher  animals,  has  a  pronounced  stimulating  action  on  the 
oxygen  uptake  of  sea-urchin  spermatozoa;  this  peculiar  effect  of 
iodoacetate  is  shared  by  other  sulphydryl-binding  compounds,  as 
well  as  by  malonate  and  nitrogen  mustard  (Barron  and  Goldinger, 
\9A\b',  Barron,  Nelson  and  Ardao,  1948;  Barron,  Seegmiller, 
Mendes  and  Narahara,  1948).  It  is  interesting  and  important  to 
note  that  widely  divergent  results  may  be  attained  with  a  given 
substance  according  to  a  particular  set  of  experimental  conditions: 
rabbit  spermatozoa,  washed  and  resuspended  in  a  sugar-free  isotonic 
medium,  are  immobilized  completely  by  00001m 2  :  3  :  5-triphenyl- 
tetrazolium  chloride,  but  a  200  times  higher  concentration  of  this 
substance  is  ineffective  towards  sperm  suspended  in  a  glucose-con- 
taining medium  (Bishop  and  Mathews,  1952).  It  is  equally  salutary 
to  bear  in  mind  that  a  substance  which  does  not  increase  the  initial 
rate  of  sperm  activity,  may  nevertheless  be  utilized  by  the  sperma- 
tozoa as  an  essential  nutrient.  For  instance,  most  of  the  sugars 
and  fatty  acids  which  are  oxidized  by  spermatozoa,  do  not  act  by 
increasing  the  initial  rate  of  respiration  but  by  maintaining  it. 

Surprisingly  enough,  many  substances  endowed  with  pronounced 
pharmocological  action  in  the  whole  animal,  such  as  the  alkaloids, 
appear  to  exert  little  or  no  effect  upon  spermatozoa  in  vitro.  Sperm 
cells  are  also  remarkably  resistant  to  ethanol.  Ivanov  (1913)  observed 
excellent  motility  in  dog  sperm  to  which  he  added  2-5%  ethanol, 
and  he  managed  to  obtain  live  and  normal  offspring  from  an  animal 
inseminated  with  semen  mixed  with  10%  ethanol.  It  may  be  men- 
tioned here  that  ethanol  is  one  of  the  substances  which  are  definitely 
known  to  pass  into  semen  after  ingestion  by  the  animal  (Farrell, 

The  Influence  of  Extraneous  Factors  57 

1938);  sulphonamides  provide  a  similar  example  (Farrell,  Lyman 
and  Youmans,  1938;  Kuehnau,  1939;  Hug,  1940). 

Marked  spermicidal  power  is  characteristic  of  a  great  many  sur- 
face-active agents,  quinones  and  heavy  metal  compounds.  A  tech- 
nique for  assessing  the  spermicidal  activity  of  pure  substances  has 
been  developed  by  Baker  (1931,  1932,  1935)  whose  so-called  'killing 
concentration'  is  the  lowest  one  capable  of  killing  all  spermatozoa 
suspended  in  a  buffered  glucose-saline  solution  (Baker's  solution) 
within  half  an  hour,  at  body  temperature,  under  standard  conditions 
in  vitro.  Of  the  many  substances  examined  by  Baker,  the  most 
highly  spermicidal  was  phenylmercuric  acetate  (killing  concentra- 
tion 0-001%).  Various  quinones  such  as  toluquinone,  butylquinone, 
methoxyquinone,  parabenzoquinone,  ethylquinone  and  paraxylo- 
quinone  were  also  strongly  spermicidal.  Lower  down  the  scale  were 
mercuric  chloride,  methoxyhydroquinone,  formaldehyde,  methyl- 
hydroquinone,  saponin,  and  hexylresorcinol.  A  critical  account  of 
the  existing  methods  for  testing  the  efficiency  of  spermicidal  com- 
pounds is  given  by  Millman  (1952),  Davidson  (1953)  and  Gamble 

As  to  the  mode  of  action  of  surface-active  agents,  such  as  e.g. 
cetyltrimethylammonium  bromide,  cetyldimethylbenzylammonium 
chloride,  /7-tri/5opropy  Iphenoxypolyethoxyethanol,  dodecyl  sulphate, 
various  condensation  products  of  long-chain  fatty  alcohols  with 
ethylene  oxide,  and  the  numerous  other  ionic  and  non-ionic  deter- 
gents, there  is  some  evidence  to  show  that  these  substances  act 
directly  on  the  constituents  of  the  so-called  lipid  capsule,  i.e.  the 
lipid-containing  outer  layer  which  protects  the  surface  of  the  sperma- 
tozoon. The  mechanism  of  the  spermicidal  activity  of  detergents  on 
the  sperm  may  be  likened  to  the  haemolytic  action  of  surface-active 
compounds  on  the  erythrocytes,  or  the  bactericidal  effects  of  these 
substances  on  various  microorganisms.  The  changes  brought  about 
by  detergents  manifest  themselves  in  a  loss  of  motility  and  fructo lytic 
power,  and  in  a  grossly  altered  permeability  of  the  sperm  cells  as 
indicated  by  the  leakage  of  cytochrome  c.  In  contrast  to  intact 
spermatozoa,  the  respiration  of  sperm  cells  treated  with  suitable 
concentrations  of  detergents  is  markedly  increased  by  succinate 
(Koefoed-Johnsen  and  Mann,  1954). 

As  to  the  action  of  some   at   any   rate,  of  the  many  other 

58  The  Biochemistry  of  Semen 

spermicidal  substances,  including  organo-metallic  compounds,  there 
are  indications  that  it  is  due  to  the  blockage  of  vital  sulphydryl 
groups  in  the  spermatozoa.  Thus  for  example,  the  immobilizing  effect 
of  iodoacetate  on  bovine  spermatozoa,  which  has  been  studied  by 
Lardy  and  Phillips  (19436),  is  most  probably  due  to  the  sulphydryl 
group-binding  capacity  of  this  substance.  In  a  war-time  study 
MacLeod  (1946Z))  showed  that  the  inhibition  of  the  metabolism  and 
motility  of  human  spermatozoa  by  organic  arsenicals  can  be  over- 
come by  1:2:  3-trithiolpropane,  and  that  the  inhibitory  effect  of 
cupric  ions  can  be  prevented  by  the  addition  of  cysteine  or  gluta- 
thione (MacLeod,  1951).  Researches  by  Mann  and  Leone  (1953) 
have  shown  that  both  motility  and  fructolysis  are  abolished  in 
mammalian  spermatozoa  by  several  thiol  reagents,  including  cupric 
ions,  hydrogen  peroxide  and  o-iodobenzoate  but  that  ergothioneine,  a 
normal  constituent  of  boar  seminal  plasma,  can  efficiently  counteract 
the  paralysing  action  of  these  reagents.  The  oxidation  of  sulphydryl 
groups  probably  explains  also  the  spermicidal  action  of  hydro- 
gen peroxide.  The  toxicity  of  hydrogen  peroxide  to  spermatozoa, 
noted  already  by  Guenther  (1907),  is  of  particular  interest  since  this 
substance  can  actually  be  formed  under  certain  conditions  by  the 
spermatozoa  themselves,  in  the  course  of  their  aerobic  metabolism, 
and  is  responsible  for  the  so-called  oxygen  damage  which  occurs 
as  a  result  of  oxygenation  of  semen  (MacLeod,  1943^;  Tosic  and 
Walton,  1946o,  b;  VanDemark,  Salisbury  and  Bratton,  1949).  Yet 
another  aspect  of  the  damaging  action  of  hydrogen  peroxide  has 
been  revealed  by  studies  on  the  effect  of  X-rays  on  spermatozoa. 
When  sea-water  heavily  irradiated  with  X-rays  was  used  as  a  diluent 
for  sea-urchin  semen,  there  was  a  great  reduction  in  the  survival 
period  of  spermatozoa  and  a  considerable  delay  in  the  cleavage  of 
the  eggs  fertilized  with  these  spermatozoa.  The  responsible  toxic 
agent  present  in  the  irradiated  sea-water  has  been  identified  by 
Evans  (1947)  as  hydrogen  peroxide.  It  is  only  fair  to  add,  however, 
that  not  all  investigators  agree  with  the  conclusion  that  the  toxicity 
of  irradiated  media  is  due  exclusively  to  hydrogen  peroxide. 

Chemical  aspects  of  short-wave  radiation 

The  chemical  changes  underlying  the  action  of  X-rays  on  sperma- 
tozoa still  remain  largely  unexplored,  and  the  precise  targets  of  this 

The  Influence  of  Extraneous  Factors  59 

and  other  forms  of  radiation  in  the  sperm  cell  are  by  no  means 
established.  The  amount  of  information  on  this  subject,  however,  is 
steadily  mounting,  ever  since  Bohn's  (1903)  and  Hertwig's  (1911) 
fundamental  observations  on  the  abnormal  development  of  sea- 
urchin  and  frog  ova  inseminated  with  spermatozoa  previously  ex- 
posed to  radium  emanation.  The  exposure  of  the  testes  to  relatively 
small  doses  of  X-ray  results  in  sterility  because  of  the  extreme 
sensitivity  of  the  seminiferous  epithelium  and  complete  breakdown 
of  spermatogenesis;  but  direct  irradiation  of  ripe,  ejaculated  sperma- 
tozoa, in  which  the  nuclei  are  in  the  resting  state,  has  usually  little 
or  no  effect  on  motility,  longevity,  morphology  or  metabolism  of 
sperm.  Nevertheless,  irradiated  spermatozoa  are  either  altogether 
infertile  or,  if  they  retain  the  power  to  penetrate  the  ovum  and  effect 
syngamy,  they  are  incapable  of  inducing  normal  development  of  the 
ovum  owing  to  damaged  chromatin.  This  conclusion  is  the  outcome 
of  extensive  investigations  on  irradiated  spermatozoa  of  several 
species,  including  sea-urchins  (Henshaw,  1940;  Barron,  Gasvoda 
and  Flood,  1949;  Blum,  1951),  frogs  (Bardeen,  1907;  Dalcq  and 
Simon,  1931;  Rugh,  1939),  insects  (Barth,  1929;  Eker,  1937),  rats 
(Henson,  1942;  Fogg  and  Cowing,  1952),  mice  (Snell,  1935),  rabbits 
(Asdell  and  Warren,  1931;  Amoroso  and  Parkes,  1947;  Murphree, 
Whitaker,  Wilding  and  Rust,  1952),  and  fowl  (Kosin,  1944).  The 
irradiation  of  cock  semen  in  vitro  with  X-ray  doses  up  to  10,000  r 
has  been  shown  by  Kosin  to  have  no  detectable  effect  on  the  motility, 
respiration  and  anaerobic  glycolysis,  but  the  fertilizing  capacity  of 
these  spermatozoa  was  markedly  reduced  already  after  exposure  to 
200  r,  and  was  altogether  destroyed  after  a  dose  of  5500  r.  These 
results  serve  to  underline  the  fact  that  sperm  fertility  may  react  to 
extraneous  factors  in  a  different  manner  from  sperm  motility  and 
metabolism.  In  our  experience,  ram  semen  irradiated  with  100,000  r 
and  examined  immediately  after  exposure,  has  normal  motility, 
fructolysis  and  adenosine  triphosphate  content;  evidently,  the  X-ray 
injury  inflicted  upon  the  spermatozoa  as  reflected  in  their  diminished 
fertilizing  capacity,  must  be  the  result  of  some  other  chemical  change, 
possibly  in  the  state  of  polymerization  of  the  deoxyribonucleic  acid 
in  the  sperm  chromatin. 

The  sensitivity  of  the  spermatogenic  tissue  to  short-wave  radiation 
is  in  marked  contrast  to  the  apparent  resistance  of  the  male  accessory 

60  The  Biochemistry  of  Semen 

glands  which  elaborate  the  seminal  plasma.  In  rats  after  total  body 
exposure  to  500  r,  there  was  occasionally  a  small  but  transient  de- 
crease in  the  level  of  fructose  and  citric  acid  secreted  by  the  acces- 
sory organs  but  in  surviving  animals  the  activity  was  restored  to 
normal  within  a  few  weeks  after  irradiation.  This  was  a  stage  when 
the  spermatozoa  were  mostly  immotile  and  a  large  proportion  of 
them  severely  damaged  (Lutwak-Mann  and  Mann,  19506). 

Variations  in  hydrogen  ion  concentration  and  tonicity 

Hydrogen  ion  concentration  is  undoubtedly  one  of  the  most 
important  factors  which  influence  the  motility,  viability  and  meta- 
bolism of  spermatozoa  in  all  species  from  sea-urchin  to  man  (Cohn, 
1917,  1918;  Wolf,  1921;  Gellhorn,  1920,  1927;  Healy  and  Anderson, 
1922;  Mettenleiter,  1925;  Barthelemy,  1926;  Yamane  and  Kato, 
1928;  Komatsu,  1929;  Schlenk,  1933;  Grodzinski  and  Marchlewski, 
1935).  Most  authors  agree  that  a  value  just  above  pH  7  provides 
the  optimum  for  the  survival  of  spermatozoa.  Sperm  respiration 
is  stated  to  be  optimal  at  the  following  pH  values,  boar  7 •2-7- 5, 
ram,  70-7-2,  bull  6-9-70,  cock  7-25,  rabbit  6-8  (Winchester  and 
McKenzie,  1941;  Lardy  and  Phillips,  1943«).  Below  the  optimum, 
motility  and  metabolism  alike  decline  progressively.  Alkalinity  on 
the  other  hand,  up  to  pH  8-5  and  above,  has  frequently  been  ob- 
served to  enhance  the  movement,  particularly  of  human  spermatozoa. 
Whereas  in  some  species  including  several  fishes,  the  spermatozoa 
are  known  to  be  extremely  sensitive  to  changes  in  pH,  in  others,  e.g. 
in  the  frog,  certain  birds  and  mammals,  they  exhibit  a  remarkable 
degree  of  resistance  (Gellhorn,  1920,  1922,  1927).  In  the  case  of 
rabbit  sperm  partially  motile  spermatozoa  have  been  found  within 
the  range  of  pH  5-0-8-8  (Cole,  Waletzky  and  Shackelford,  1940). 
More  recently  Emmens  (1947)  has  shown  that  even  at  pH  9-5-10-0 
rabbit  spermatozoa  retain  partial  motility  for  several  hours,  but 
they  become  immotile  and  die  rapidly  at  pH  values  below  5-8. 
According  to  this  author,  the  point  at  which  the  progressive  move- 
ment is  abolished  and  motility  reduced  to  a  condition  where  heads 
become  completely  stationary  but  tails  still  retain  feeble  motion, 
coincides  with  a  state  when  about  50%  of  the  sperm  cells  can  be 
shown  to  be  dead  by  the  differential  staining  method  of  Lasley, 
Easley  and  McKenzie  (1942);  in  this  method,  dead  spermatozoa  take 

The  Influence  of  Extraneous  Factors  61 

up  eosin  whereas  live  cells  remain  unstained.  The  time  required  to 
reach  the  50%  mortality  level  was  stated  to  be  6  hr.  at  pH  6-4-6-5, 
29  hr.  at  pH  7-2-7-9,  15  hr.  at  pH  8-5-9-5,  7  hr.  at  pH  9-7-9-8,  and 
4  hr.  30  min.  at  pH  10-2. 

However,  even  after  spermatozoa  have  been  rendered  immotile 
by  excessive  acidity  they  can  still  be  resuscitated  by  alkalinization, 
always  provided  that  the  exposure  to  acid  has  not  been  unduly 
long(Engelmann,  1868;  Lillie,  1913, 1919;  Gray,  1915;  Muschat,  1926; 
Schlenk,  1933).  Under  conditions  in  vitro,  the  time  intervals  at  which 
the  sperm  can  be  revived,  correlate  well  with  the  mortality  rate 
(Emmens,  1947);  no  reactivation  was  seen  when  the  death  rate  of 
rabbit  sperm  exceeded  80%. 

The  slowing  down  effect  of  weak  acids  and  the  reactivating  in- 
fluence of  weak  alkalis  may  well  be  of  some  importance  for  the 
activity  of  spermatozoa  in  vivo,  in  the  various  parts  of  the  male  and 
female  reproductive  tract.  There  are,  however,  several  other  im- 
portant factors  which  influence  sperm  motility  and  survival  in  vivo, 
such  as  the  concentration  of  various  ions  and  nutrients,  dilution, 
and  the  tension  of  oxygen.  Thus,  in  the  epididymis,  and  partly  also 
in  the  vas  deferens,  the  spermatozoa  are  immotile  at  a  pH  which 
approximates  neutrahty  (Lanz,  1929;  Bishop  and  Mathews,  1952); 
here,  presumably,  the  combination  of  the  very  low  oxygen  tension, 
deficiency  of  carbohydrate  and  limitation  of  space  acquires  greater 
significance  than  the  hydrogen  ion  concentration. 

Tonicity  as  a  sperm-affecting  factor  ranks  equal  in  importance 
with  the  hydrogen  ion  concentration.  Most  investigators  of  semen, 
including  Yamane  (1920),  Gellhorn  (1922,  1924,  1927),  and  Milo- 
vanov  (1934),  agree  that  on  the  whole,  spermatozoa  seem  to  be 
immobilized  much  more  readily  by  hypotonic  than  hypertonic, 
diluents.  However,  it  must  be  remembered  that  the  ultimate  effect  of 
tonicity  depends  upon  certain  other  prevailing  conditions.  Thus, 
for  example,  Emmens  (1948)  who  studied  the  motility  of  rabbit 
spermatozoa  at  various  pH  values  with  diluents  of  different  chemical 
composition  and  tonicity,  has  shown  that  at  pH  5  •8-6-6,  the  sperma- 
tozoa were  more  sensitive  to  hypotonicity  than  to  hypertonicity,  but 
in  an  alkaline  medium  the  situation  was  reversed.  In  an  analogous 
study  of  ram,  bull  and  human  spermatozoa  (Blackshaw  and  Emmens, 
1951)  it  was  established  that  at  all  pH  levels,  hypertonic  solutions 

62  The  Biochemistry  of  Semen 

were  less  harmful  to  motility  than  hypotonic  media,  and  that  the 
relatively  slight  adverse  effect  of  hypertonicity  could  be  diminished 
by  partial  replacement  of  sodium  chloride  with  glucose.  Further- 
more, the  extent  to  which  a  hypotonic  or  hypertonic  medium  can 
affect  the  spermatozoa,  very  much  depends  on  the  degree  of  sperm 
dilution.  Highly  concentrated  solutions  of  sodium  chloride  incor- 
porated in  so-called  salt-jellies,  have  been  shown  to  possess  marked 
spermicidal  properties  (Gamble,  1953). 

Influence  of  heat  and  cold;  sperm  vitrification  and  'la  vie  latente'' 

Temperature  has  long  been  known  to  exert  a  powerful  influence  in 
determining  the  onset  of  spermatogenesis  and  breeding  activity  in 
animals.  The  effect  of  increased  temperature  on  the  male  reproduc- 
tive organs  presents  many  intriguing  questions.  Hyperpyrexia  fre- 
quently causes  a  temporary  azoospermia  in  man,  and  a  hot  climate 
is  believed  to  be  the  principal  cause  of  certain  forms  of  subfertility 
among  domestic  animals  in  tropical  countries.  Mammalian  testes 
removed  from  the  scrotum  and  placed  in  the  abdominal  cavity, 
where  the  temperature  is  a  few  degrees  higher,  cease  to  produce 
spermatozoa;  degeneration  of  the  spermatogenic  tissue  sets  in  and 
spermatogenic  function  is  not  resumed  while  'experimental  crypt- 
orchidism' prevails.  In  the  guinea-pig,  a  complete  cessation  of  sper- 
matogenesis can  be  brought  about  experimentally  by  scrotal  appli- 
cation of  heat  (6°  above  the  normal  body  temperature)  for  a  period 
of  10  min.  (Moore,  1924,  1951).  A  similar  effect  can  be  produced 
in  rams;  semen  collected  from  such  animals  a  week  or  two  later 
contains  only  a  small  number  of  spermatozoa,  mostly  dead  or 
degenerate;  the  seminal  plasma  on  the  other  hand,  retains  its 
normal  composition  or  shows  even  a  slightly  elevated  content  of 
fructose  (Glover,  1954).  In  bulls,  heat-induced  azoospermia  is  said 
to  be  associated  with  an  increased  excretion  of  neutral  steroids  in 
the  urine  (Meschaks,  1953).  Cold,  like  heat,  has  an  adverse  effect  on 
sperm  cells  in  vivo.  An  ice-pack  applied  for  10  min.  to  the  testes 
of  a  rabbit  invariably  results  in  disintegration  of  spermatozoa  in 
the  epididymides  (Chang,  1943). 

No  less  dramatic  but  different  in  kind  are  the  in  vitro  effects  of 
heat  and  cold  on  spermatozoa.  Dog  and  rabbit  spermatozoa  although 
capable  of  survival  for  several  hours  in  vitro  at  40°,  soon  lose  their 

The  Influence  of  Extraneous  Factors  63 

motility  at  45°  (Amantea  and  Krzyszkowsky,  1921;  Walton,  1930). 
Cooling  to  a  temperature  just  above  0°  is  not  harmful  to  ejaculated 
semen  in  vitro,  provided  however,  that  the  temperature  of  the 
ejaculate  has  been  lowered  gradually,  preferably  by  successive 
stages  of  5°,  with  an  interval  of  2  hr.  or  so,  between  each.  Sudden 
cooling  of  ejaculated  semen  produces  so-called  temperature  shock 
and  involves  rapid  and  irreversible  loss  of  motility  and  fertilizing 
power  (Gladcinova,  1937;  Chang  and  Walton,  1940;  Easley,  Mayer 
and  Bogart,  1942).  The  decline  of  respiration  and  fructolysis  in 
'temperature-shocked'  samples  of  bull  semen  was  shown  to  be  cor- 
related with  a  proportionate  increase  of  dead,  that  is  eosin-staining, 
sperm  cells  (Hancock,  1952). 

Provided  that  strict  precautions  are  observed,  semen  can  be  cooled 
well  below  0°  without  destroying  the  sperm.  Some  of  the  earliest 
observations  concerning  the  effect  of  low  temperature  on  sperm 
(human,  bull,  stallion,  and  frog)  were  made  by  Spallanzani  (1776, 
1799).  On  subjecting  stallion  spermatozoa  'to  the  cold  of  freezing, 
by  putting  the  glass  in  which  they  were,  among  snow'  he  made  the 
following  observations: 

'The  same  effect  was  produced  by  snow,  as  by  the  winter's  cold; 
that  is,  in  fourteen  minutes,  it  made  the  spermatozoa  motionless; 
although  when  exposed  to  the  heat  of  the  atmosphere,  they  con- 
tinued to  move  seven  hours  and  a  half.  But  an  accident  that  hap- 
pened in  this  experiment,  executed  during  summer,  afforded  new 
intelligence,  and  divested  me  of  a  prejudice.  Observing  that  the 
vermiculi  had  become  motionless,  I  took  the  glass  from  the  snow, 
and  left  it  exposed  to  the  air,  when  the  heat  was  27°.  An  hour  after, 
by  chance  observing  this  semen  with  the  microscope,  I  was  astonished 
to  find  all  the  vermiculi  reanimated,  and  in  such  a  manner,  as  if 
they  had  just  come  from  the  seminal  vessels.  I  then  saw,  that  the 
cold  had  not  killed  them,  but  had  reduced  them  to  a  state  of  com- 
plete inaction.  I  replaced  them  in  the  snow,  and  in  three  quarters  of 
an  hour  took  them  away.  These  are  the  phenomena  I  observed.  In 
a  few  minutes,  their  vivacity  relaxed,  and  the  diminution  increased, 
until  they  lost  the  progressive  motion,  and  retained  only  that  of  oscil- 
lation, which  likewise  ended  in  a  few  minutes  more.  Exactly  the 
reverse  was  observed,  when  they  passed  from  the  cold  of  the  snow  to 
the  heat  of  the  atmosphere.  The  first  motion  that  appeared,  was 
that  of  oscillation;  the  body  and  the  tail  begun  to  vibrate  languidly 

64  The  Biochemistry  of  Semen 

from  right  to  left;  then  the  motion  was  communicated  to  the  whole 
vermicule;  and,  in  a  short  time,  the  progressive  motion  begun.' 

In  the  XlXth  century,  Prevost  (1840),  de  Quatrefages  (1853), 
Mantegazza  (1866),  Schenk  (1870)  and  others,  experimented  with 
sperm  exposed  to  temperatures  ranging  from  0°  to  -17°,  but  it 
was  not  until  1938  when  Jahnel  proved  that  human  spermatozoa 
can  resist  the  temperature  of  solid  carbon  dioxide  (-79"),  and  Luyet 
and  Hodapp  demonstrated  that  frog  spermatozoa  can  survive  the 
temperature  of  liquid  air  (-192°),  provided  that  they  are  mixed  with 
a  concentrated  solution  of  sucrose  before  immersion  in  liquid  air. 

These  and  subsequent  studies  by  other  authors,  including  Shettles 
(1940),  Shaffner  (1942),  Hoagland  and  Pincus  (1942)  and  Parkes 
(1945)  have  provided  further  strong  support  for  the  general  con- 
clusion, elaborated  in  detail  by  Luyet  and  Gehenio  (1940)  in  their 
treatise  on  Life  and  Death  at  Low  Temperatures,  namely  that  sperma- 
tozoa, not  unlike  certain  bacteria  and  some  flagellates,  are  remark- 
ably resistant  to  low  temperatures  and  on  vitrification  pass  into  a 
reversible  condition  of  complete  inactivity  and  quiescence.  This 
was  described  by  Becquerel  as  ia  vie  latente'  and  has  been  compared 
to  the  behaviour  of  a  watch  which,  though  well  wound,  can  be 
brought  to  a  sudden  standstill  by  some  braking  mechanism;  such  a 
watch  will  start  of  its  own  accord  as  soon  as  the  brake  is  removed. 
The  main  principle  underlying  Luyet's  thesis  is  that  cells  such  as 
spermatozoa  manage  to  survive  at  low  temperatures  if  cooling  is 
effected  so  as  to  by-pass  the  crystallization  zone,  by  carrying  the  cells 
straight  into  the  range  of  sub-freezing  temperatures  known  as  the 
vitrification  zone,  where  they  assume  the  non-crystalline,  glass-like, 
or  vitreous  state.  The  passage  on  thawing  from  the  vitreous  state 
equally  deserves  attention  and  is  best  achieved  by  rapid  warming, 
again  to  avoid  the  crystallization  zone.  When  these  precautions  are 
maintained,  it  is  possible  to  prevent  colloidal  changes  ordinarily 
associated  with  freezing  and  ice-crystal  formation,  such  as  dena- 
turation  and  coagulation  of  proteins,  protoplasmic  precipitation, 
release  of  enzymes  and  structural  disarrangement.  In  the  opinion 
of  Becquerel  (1936,  1938),  the  principal  danger  to  the  'latent  life' 
of  cells  at  low  temperatures  is  the  damage  to  cellular  structure 
which  occurs  in  the  freezing  zone,  caused  by  the  separation  of  water 

The  Influence  of  Extraneous  Factors  65 

and  electrolytes  from  the  colloidal  particles.  This  damage  can  be 
substantially  reduced  if  freezing  is  carried  out  in  the  presence  of 
certain  organic  substances  such  as  sucrose,  glucose,  fructose, 
glycerol,  ethylene  glycol,  gelatin,  albumin  and  various  gums,  all  of 
which  have  been  used  extensively  in  the  past  in  freezing  and  freeze- 
drying  experiments  on  bacteria,  yeasts,  protozoa  and  various  other 
cells  and  tissues.  The  application  of  these  substances,  however,  to 
sperm  is  comparatively  recent.  In  1938,  Luyet  and  Hodapp  ob- 
served that  frog  spermatozoa  fail  to  survive  the  temperature  of 
liquid  air,  but  if  the  cooling  is  carried  out  in  the  presence  of  40% 
sucrose  at  least  20%  of  them  revive.  Shaffner,  Henderson  and  Card 
(1941)  were  able  to  keep  alive  30%  of  fowl  spermatozoa  by  freezing 
them  to  -79°,  after  treatment  with  fructose.  An  attempt  to  use 
glycerol  in  connection  with  survival  experiments  on  frog  sperma- 
tozoa was  made  by  Rostand  (1946)  but  it  was  not  until  1949  when 
the  remarkable  properties  of  glycerol  were  brought  into  prominence 
thanks  to  Polge,  Smith  and  Parkes  (1949)  as  a  result  of  their  studies 
on  the  low  temperature  resistance  of  glycerol-treated  fowl  semen.  In 
fowl  semen  diluted  with  an  equal  volume  of  Ringer  solution  and 
vitrified  at  -79°  for  20  min.  and  then  rapidly  thawed,  there  was  no 
significant  revival  of  spermatozoa.  On  the  other  hand,  when  the 
dilution  was  carried  out  with  Ringer  solution  containing  40% 
glycerol,  the  spermatozoa  resumed  full  motility  on  thawing.  This 
observation  was  soon  extended  to  the  semen  of  other  animals, 
including  the  bull  (Smith  and  Polge,  1950).  A  large  number  of  cows 
have  been  inseminated  by  Polge  and  Rowson  (1952)  with  glycerol- 
treated  bull  semen  which  had  been  stored  at  -79°  for  periods  of 
many  months,  and  the  excellent  fertilizing  capacity  of  such  'deep- 
frozen'  semen  was  proved  when  pregnancy  occurred  in  66%  of  the 
inseminated  animals.  Glycerol-frozen  and  thawed  human  semen  has 
been  reported  to  contain  motile  and  fertile  spermatozoa  (Sherman 
and  Bunge,  1953;  Bunge  and  Sherman,  1953). 

As  yet,  there  is  no  adequate  explanation  for  the  effect  of  glycerol 
on  semen.  The  suggestion  that  it  acts  by  supporting  some  sort  of 
residual  metabolism  in  the  frozen  spermatozoa  is  difficult  to  reconcile 
with  our  own  observation  that  glycerol,  unlike  sugars  and  fatty  acids, 
is  not  oxidized  by  bull  or  ram  spermatozoa.  More  probably,  glycerol 
exerts  a  protective  influence  on  spermatozoa,  preventing  denaturation 

66  The  Biochemistry  of  Semen 

changes  during  freezing.  Glycerol  has  long  been  recognized  in 
protein  and  enzyme  chemistry  as  a  convenient,  'stabilizing'  agent 
which  combines  the  properties  of  a  protein  solvent  with  the  ability 
to  protect  the  protein  from  denaturation  caused  by  temperature 
changes.  It  has  been  shown  to  prevent  the  heat  coagulation  of  serum 
and  egg  albumin  (Beilinsson,  1929),  and  is  in  use  in  the  cold  storage 
of  egg  yolk  owing  to  its  solubilizing  action  on  lipoproteins  (cf. 
McFarlane  and  Hall,  1943).  Lavin,  Northrop  and  Taylor  (1935) 
used  glycerol  in  their  study  of  pepsin  at  -100°;  Keilin  and  Hartree 
(1949)  discovered  that  the  presence  of  glycerol  at  very  low  tempera- 
tures intensifies  the  absorption  spectra  of  haemoproteins  some 
twenty-five  times.  This  in  turn,  made  it  possible  to  demonstrate  for 
the  first  time,  the  spectrum  of  cytochrome  in  human  spermatozoa 
(Mann,  1951^).  The  protecting  influence  of  glycerol  on  sperm  col- 
loids may  well  be  linked  with  the  electrolyte-  and  water-binding 
properties  of  this  substance.  In  this  connection,  an  observation  by 
Luyet  deserves  to  be  mentioned,  on  the  existence  of  a  definite  rela- 
tionship between  the  water-binding  capacity  of  different  solutes 
used  for  vitrification,  and  the  temperature  at  which  devitrification 
takes  place  on  thawing;  the  devitrification  temperature  of  glycerol 
is  in  the  neighbourhood  of  -70°  (Miner  and  Dalton,  1953)  which  is 
below  that  of  sucrose,  fructose,  gelatin,  various  gums  and  most 
other  solutes. 

Role  of  hormones 

Among  factors  which  influence  the  production  of  semen  in  man 
and  animals,  hormones  rank  paramount  in  importance.  The  forma- 
tion, output,  and  composition  of  ejaculated  semen  are  the  outcome 
of  a  concerted  action  of  several  endocrine  organs,  with  the  pituitary 
gland  and  the  testicular  interstitial  tissue  in  dominant  positions. 

Apart  from  the  direct  gonadotrophic  activity  due  to  the  game- 
togenic  and  interstitial-cell  stimulating  hormones,  the  anterior  lobe 
of  the  pituitary  gland  exerts  an  indirect  influence  upon  the  male 
reproductive  organs,  through  interaction  with  the  thyroid  gland  and 
the  adrenal  cortex.  The  anterior  hypophysis  itself  depends  on  stimuli 
from  the  central  nervous  system  and  responds  in  a  particularly  sen- 
sitive manner  to  impulses  transmitted  through  the  optic  nerves. 
The  seasonal  fluctuations  in  the  intensity  of  light  impulses,  relayed 

The  Influence  of  Extraneous  Factors  67 

through  the  optic  nerves,  are  probably  responsible  for  the  changes 
associated  with  the  so-called  male  sex  cycle;  many  lower  vertebrates 
produce  spermatozoa  only  during  a  brief  period,  once  a  year,  and 
even  among  mammals,  there  are  many  species  in  which  male  sexual 
activity  is  restricted  to  definite  season.  Sperm  survival  in  the  epidi- 
dymis and  sperm  transport  in  the  female  reproductive  tract,  are  also 
two  phenomena  which  probably  depend  on  pituitary  function;  the 
former  on  the  normal  gonadotrophic  activity  of  the  anterior  lobe, 
the  latter  on  impulses  transmitted  to  the  uterus  by  the  oxytocic 
hormone  secreted  in  the  posterior  lobe. 

The  function  of  testicular  hormone,  in  so  far  as  semen  is  con- 
cerned, is  to  provide  the  stimulus  necessary  for  the  elaboration  of 
seminal  plasma  and  for  ejaculation.  The  male  sex  hormone  is 
intrinsically  linked  with  the  production  of  seminal  plasma  by  the 
accessory  organs;  it  regulates  the  secretory  activity  of  the  accessory 
organs,  and  thus  determines  not  only  the  output  of  the  seminal 
plasma  as  a  whole,  but  also  the  relative  contribution  of  each  indi- 
vidual gland  towards  the  ultimate  make-up  of  semen. 

The  earlier  morphological  studies  have  provided  much  funda- 
mental information  which  has  helped  to  build  up  our  knowledge  of 
the  relationship  between  the  functional  state  of  the  male  accessory 
organs  and  androgenic  activity.  These  investigations  have  shown 
that  postcastrate  retrogressive  changes  in  the  gross  appearance  and 
in  the  microscopic  structure  of  the  accessory  organs  can  be  pre- 
vented, or  reversed,  by  the  administration  of  testicular  hormone.  On 
this  basis  several  so-called  'hormone  indicator  tests'  have  been 
elaborated  for  the  detection  of  male  sex  hormone  activity  (cf.  Moore, 
1937;  Price,  1947;  Dorfman,  1950).  Two  such  tests  also  involved 
semen  examination:  the  so-called  'clotting  test'  was  based  on  the 
observation  that  the  formation  of  a  clot  in  the  electrically-induced 
seminal  discharge  of  a  guinea-pig  depends  on  the  presence  of  male 
sex  hormone,  and  the  'spermatozoon  motility  test'  was  derived 
from  the  observation  that  in  the  epididymis  severed  from  the  testis, 
spermatozoa  survive  longer  if  the  testis  is  not  removed  from  the 
body;  presumably,  the  testicular  hormone  is  capable  of  stimulating 
the  epididymal  cells  to  secrete  some  substance  necessary  for  the 
preservation  of  epididymal  spermatozoa  (Moore,  1935;  Parsons, 

68  The  Biochemistry  of  Semen 

Of  recent  years  the  introduction  of  biochemical  methods  made  it 
practicable  to  follow  up  and  to  assess  androgenic  or  gonadotrophic 
activity,  or  changes  due  to  hormonal  deficiency,  by  means  of  quanti- 
tative chemical  analysis  of  the  seminal  plasma.  Determinations  of 
fructose,  citric  acid  and  phosphatase  activity  in  semen  provide  excel- 
lent evidence  of  the  functional  state  of  the  male  accessory  organs  of 
reproduction.  These  methods  which  will  be  discussed  in  detail  later, 
are  particularly  useful  in  studies  of  progressive  hormonal  deficiency, 
such  as  is  brought  about  for  instance,  by  defective  nutrition.  The 
great  advantage  of  the  chemical  approach  is  that  it  enables  the 
assay  of  accessory  gland  function  to  be  carried  out  in  live  animals, 
at  selected  intervals,  and  over  long  periods  of  time. 

So  far,  there  has  been  little  progress  in  investigations  concerning 
the  influence  of  hormones  on  semen  in  vitro.  Several  hormones  have 
been  variously  credited  with  beneficial  effects  upon  the  survival, 
motility  and  metabolism  of  sperm  in  vitro  (cf.  Tschumi,  1946),  but 
in  actual  fact,  apart  from  isolated  observations  such  as  those 
concerning  the  stimulating  effect  of  thyroxine  on  spermatozoa 
(Carter,  1931,  1932;  Lardy  and  Phillips,  1943^;  Schultze  and  Davis, 
1948,  1949;  Maqsood,  1952),  the  evidence  at  hand  requires  much 
strengthening  before  the  various  claims  are  accepted  as  valid.  The 
same  is  true  of  studies  on  the  content  of  hormones  in  semen  itself. 
There  are  indications  that  semen  contains  some  oestrogenic  sub- 
stances (Green- Army tage  et  al.,  1947;  McCullagh  and  Schaffenburg, 
1951;  Mukherjee  et  al.,  1951)  which  is  not  improbable  since  oestro- 
genic hormones  occur  elsewhere  in  the  male  body,  notably  in  the 
testis  and  in  the  urine.  Diczfalusy  (1954)  examined  by  counter- 
current  distribution  and  fluorimetric  analysis  an  alcoholic  extract 
from  a  litre  of  human  semen  and  found  in  this  material  10  fig.  of 
oestradiol-lT/S,  30  jug.  oestriol,  and  60  jug.  oestrone,  all  in  a  free 
non-conjugated  form. 

With  respect  to  androgens,  the  Dirscherl-Zilliken  colour  reaction 
for  dehydro/56>androsterone  is  strongly  positive  in  extracts  from 
hydrolysed  spermatozoa.  According  to  Dirscherl  and  Kniichel 
(1950),  the  content  of  the  colour-yielding  material  corresponds  to 
about  5-5  mg.  'dehydroandrosterone'  in  100  ml.  human  semen,  as 
compared  with  0-1  mg./lOO  ml.  in  human  urine;  for  bull  and  stallion 
semen  the  values  are  given  as  4-3  and  1-6  mg./lOO  ml.,  respectively. 

The  Influence  of  Extraneous  Factors  69 

The  bulk  of  the  androgenic  material  appears  to  occur  in  a  bound 
form  and  can  be  set  free  by  hydrolysis  with  hydrochloric  acid. 

c    I    c 

QIX  13C  \ 

18CH3 1  I  leC 

C  C         14C  / 

/^\   /.\   /   \ia/ 

Q2  QIO  8C  C 

C3  5C  7C 

c        c 

The  tetracyclic  carbon  skeleton 
in  androgens 









Sperm-egg  interacting  substances  and  chemotaxis 

The  so-called  sperm-egg  interacting  substances  comprise  a  group 
of  agents  which  have  received  considerable  attention  from  pioneers 
in  the  field  of  sex  physiology  such  as  Lillie  (1919)  and  Loeb  (1913), 

70  The  Biochemistry  of  Semen 

but  have  acquired  even  more  prominence  and  a  wider  significance  in 
recent  times,  chiefly  as  the  outcome  of  investigations  by  the  schools 
of  Hartmann  in  Germany,  Runnstrom  in  Sweden,  and  Tyler  in  the 
United  States.  So  far,  research  on  these  substances  has  been  Umited 
largely  to  invertebrates;  it  has  been  thoroughly  reviewed  by  Tyler 
(1948),  Bielig  and  von  Medem  (1949),  Runnstrom  (1949,  1951), 
Brachet  (1950)  and  Rothschild  (1951^,  b)  and  will  therefore  be 
mentioned  here  only  briefly. 

The  fundamental  observations  on  sperm-egg  interacting  substances 
were  made  by  Frank  Lillie  who  discovered  that  the  so-called  'egg- 
water',  that  is  sea-water  with  which  sea-urchin  eggs  have  remained  in 
contact  for  a  while,  is  enriched  with  some  substances  derived  from 
the  eggs,  and  capable  of  inducing  the  agglutination  of  homologous 
spermatozoa.  Lillie  was  convinced  that  the  sperm-agglutinating 
agent  plays  a  significant  role  in  fertilization  and  called  it  'fertilizin'. 
Another  effect  produced  by  egg-water,  which  he  noted  in  certain 
species  of  marine  animals,  was  an  increase  in  the  motility  of  sperma- 
tozoa. This  phenomenon  he  also  ascribed  to  'fertilizin';  the  sub- 
stance in  the  sperm,  with  which  fertilizin  was  believed  to  combine, 
was  named  'antifertilizin'. 

The  sperm-egg  interacting  substances,  mostly  studied  in  sea- 
urchins,  are  sometimes  referred  to  as  'gamones';  those  derived  from 
eggs  being  called  'gynogamones',  as  opposed  to  'androgamones'  in 

Gynogamone  I  is  the  name  given  to  the  agent  responsible  for 
the  activating  influence  of  egg- water  on  spermatozoa.  Hartmann, 
Schartau,  Kuhn  and  Wallenfels  (1939)  thought  that  in  the  case  of 
Arbacia  pustulosa  the  sperm-activating  substance  is  chemically  re- 
lated to  echinochrome,  the  pigment  discovered  in  1885  by  MacMunn. 
They  found  that  pure  echinochrome  A  (2-ethyl-3  :  5  :  6  :  7  :  8-penta- 
hydroxynaphtoquinone)  isolated  from  ripe  eggs  oi  Arbacia  pustulosa, 
exerts  a  stimulating  effect  on  the  movements  of  sea-urchin  sperm, 
even  in  a  1  :  2,500,000,000  dilution.  This  claim,  however,  has  been 
seriously  challenged  by  Tyler  (1939)  and  Cornman  (1940,  1941). 

At  this  point  it  may  be  relevant  to  recall  two  observations,  one 
by  Clowes  and  Bachman  (1921^,  b)  who  noted  that  a  sperm- 
stimulating  agent  can  be  separated  from  egg-water  by  distilla- 
tion, and  the  other  a  finding  made  by  Carter  (1931)  that  the 

The  Influence  of  Extraneous  Factors  71 

activation  effect  of  egg-water  on  spermatozoa  can  be  reproduced  by 

Gynogamone  II,  also  called  'isoagglutinin',  is  the  name  applied 
to  the  fertilizin  responsible  for  the  agglutinating  action  of  egg-water 
upon  spermatozoa  of  the  same  species.  This  substance  originates  in 
the  gelatinous  material  or  'jelly  coat'  surrounding  the  eggs,  and 
passes  therefrom  into  the  sea-water;  chemically  it  is  a  mucoprotein 
which  differs  in  composition  according  to  species;  the  polysac- 
charide component  has  been  reported  to  contain  sulphuric  acid  in 
addition  to  galactose,  fucose,  glucose,  or  fructose  (Vasseur,  1947, 
1949;  Tyler,  1948;  Bishop  and  Metz,  1952). 

The  group  of  androgamones  comprises  three  substances.  Andro- 
gamone  I,  to  which  further  reference  will  be  made  later  in  connec- 
tion with  its  alleged  role  as  a  sperm-immobilizing  agent,  antagonizes 
the  action  of  gynogamone  I  (Hartmann,  Schartau  and  Wallenfels, 
1940).  It  is  a  heat-stable,  alcohol-soluble  factor  which  can  be  ex- 
tracted from  sea-urchin  spermatozoa  by  sharp  centrifugation  or 
with  methanol,  but  it  is  still  uncertain  whether  or  not  it  actually 
diffuses  out  of  intact  sperm  cells.  Androgamone  II  is  the  antifer- 
tilizin  which  reacts  with  the  sperm-agglutinating  gynagamone  11. 
It  is  an  alcohol-insoluble,  protein-like  substance  extracted  by  Hart- 
mann and  his  associates  from  sea-urchin  sperm,  and  believed  to 
function  as  a  jelly-coat  dissolving  or  precipitating  factor.  Andro- 
gamone III,  also  known  as  the  'egg-surface  liquefying  agent'  or 
'lysin',  is  an  alcohol-soluble  substance  found  by  Runnstrom,  Lind- 
vall  and  Tiselius  (1944)  in  sea-urchin  and  salmon  spermatozoa,  with 
a  lytic  action  towards  the  cortical  layer  of  eggs.  It  is  probably  a 
fatty  acid;  in  its  effect  on  sea-urchin  eggs  it  resembles  bee  venom 
and  certain  detergents  (Runnstrom  and  Lindvall,  1946). 

Much  confusion  in  the  past  has  been  caused  by  conflicting  reports 
which  ascribed  to  gynogamones,  apart  from  their  activating  or 
agglutinating  action,  also  a  definite  attracting  or  chemotactic  influ- 
ence on  spermatozoa.  It  is  doubtful  whether  in  animals  chemo taxis 
plays  any  serious  role  in  guiding  the  spermatozoa  to  the  eggs. 
Plants,  on  the  other  hand,  provide  several  excellent  examples  for 
the  existence  of  chemotaxis  (Cook,  1945;  Hawker,  1951).  In  mosses 
and  ferns,  the  spermatozoids  are  well  known  to  be  attracted  towards 
various  substances,  for  example  malic  acid,  sucrose,  certain  salts 

72  The  Biochemistry  of  Semen 

and  alkaloids;  in  a  few  instances,  some  of  these  substances  have 
actually  been  claimed  to  occur  in  the  archegonia  (Pfeffer,  1884; 
Shibata,  1911).  Bracken  spermatozoa  are  attracted  by  the  cis  but 
not  the  trans,  configuration  of  organic  acids;  thus,  they  can  be 
shown  to  move  towards  maleic  but  not  fumaric,  and  towards  citra- 
conic  but  not  mesaconic,  acid  (Rothschild,  1952).  Several  organic 
compounds,  including  some  simple  hydrocarbons,  ethers  and  esters, 
have  been  shown  to  possess  chemotropic  activity  for  the  sperm  of 
certain  Fucaceae  (Cook,  Elvidge  and  Heilbron,  1948;  Cook  and 
Elvidge,  1951). 

Cross-poUination  between  two  flowering  plants  of  Forsythia  is 
brought  about  by  an  exchange,  followed  by  enzymic  breakdown,  of 
two  glycosides  of  the  natural  flavonol  pigment  quercetin,  carried 
with  the  pollen,  namely  rutin  (quercetin  rutinoside)  and  quercitrin 
(quercetin  rhamnoside)  (Kuhn  and  Low,  1949;  Moewus,  1950). 
Plants  also  provide  several  instances  of  difierential  distribution  of 
pigments  in  the  male  and  female  gametes.  Among  the  fungi,  the 
small  motile  male  gamete  of  some  species  of  Allomyces  is  distin- 
guished from  the  larger  female  gamete  by  the  presence  of  an  orange- 
coloured  globule  in  which  Emerson  and  Fox  (1940)  have  found 
y-carotene  along  with  traces  of  isomers.  In  the  family  Fucaceae, 
chemical  resolution  of  the  pigments  from  the  male  and  female 
exudates  of  several  species  has  shown  that  the  predominant  colour- 
ing matter  of  the  orange-coloured  male  gametes  consists  of  /^-caro- 
tene,  whereas  the  olive-green  pigmented  eggs  contain  chlorophyll 
and  fucoxanthin  (Carter,  Cross,  Heilbron  and  Jones,  1948).  The 
participation  of  carotenoids  in  the  reproduction  of  algae  is  indi- 
cated by  studies  on  the  unicellular  flagellate  alga,  Chlamydomonas, 
where  picrocrocin,  crocin,  and  cis-trans-crocQtm  dimethyl  esters 
have  been  shown  to  play  a  role  in  the  conjugation  of  the  male  and 
female  gametes  as  well  as  in  sex  determination  (Kuhn,  Moewus  and 
Jerchel,  1938;  Kuhn  and  Moewus,  1940). 

In  animals,  the  position  of  carotenoids  as  sperm-active  sub- 
stances remains  uncertain,  but  there  is  the  significant  fact  that 
remarkably  large  amounts  of  carotenoid  pigments  occur  in  male 
gonads  and  accessory  glands  of  reproduction  of  many  animals, 
including  mammals  (Goodwin,  1950).  The  importance  of  vitamin  A 
and  carotene  in  developing  and  maintaining  the  normal  germinal 

The  Influence  of  Extraneous  Factors  73 

epithelium  in  bulls  has  been  stressed  repeatedly  by  many  investi- 
gators (cf.  Bratton  et  ai,  1948). 

'Dilution  effect'  and  chemical  changes  associated  with  sperm  senescence 

The  changes  which  result  from  dilution  of  semen  have  been  the 
subject  of  much  study;  two  distinct  lines  of  research  are  recognizable. 
The  chief  endeavour  of  the  mammaUan  semen  investigators  was  to 
solve  the  practical  problem  of  the  composition  of  artificial  diluents, 
whereas  the  workers  interested  in  the  sperm  of  lower  forms,  such 
as  the  sea-urchin,  were  trying  to  establish  the  cause,  rather  than  the 
remedy,  for  the  effect  of  dilution.  It  may  be  said  at  once  that  as  yet, 
there  is  no  perfect  semen  diluent,  and  the  precise  mechanism  of  the 
'dilution  effect'  still  remains  to  be  solved  but  much  progress  has 
been  made  in  both  directions. 

Some  of  the  early  experiments  on  semen  dilution,  by  Koelliker 
(1856),  Ankermann  (1857),  Engelmann  (1868),  and  others,  were 
carried  out  with  frog  spermatozoa;  in  semen  pressed  out  directly 
from  the  frog  testis,  the  spermatozoa  were  found  to  be  motionless 
but  when  mixed  with  a  few  parts  of  water,  they  became  intensely 
motile.  This  activity,  however,  was  of  short  duration;  it  began  to 
decline  already  after  a  few  minutes  and  seldom  extended  beyond  one 
hour.  Prolonged  motility  was  obtained  when  water  was  replaced 
with  0-25  to  0-5%  NaCl  solutions.  In  the  presence  of  higher  concen- 
trations of  NaCl  the  spermatozoa  remained  motionless  but  could  be 
'revived',  even  after  a  relatively  long  time,  by  further  addition  of 
water.  It  was  also  shown  that  the  presence  of  oxygen  is  not  absolutely 
essential  for  the  motihty  of  frog  spermatozoa.  This  was  first  demon- 
strated in  1868  by  Engelmann  at  Utrecht  who  found  that  frog  sperm 
motility  could  be  maintained  for  several  hours  in  diluted  semen,  in 
an  atmosphere  of  hydrogen  or  carbon  monoxide;  however,  when  this 
'anaerobic'  motility  began  to  decline,  it  was  restored  by  pure  oxygen 
or  air. 

The  response  of  fish  and  sea-urchin  spermatozoa  to  dilution  with 
water  or  salt  solutions  is  not  very  different  from  that  of  frog  sperm. 
The  addition  of  water  or  dilute  salt  solutions  to  trout  semen  pro- 
vokes a  shortlived  burst  of  activity  followed  by  gradual  exhaustion 
and  death  of  the  spermatozoa;  oxygen  has  an  activating  effect  on 

74  The  Biochemistry  of  Semen 

the  motility  and  prolongs  the  life  of  the  spermatozoa  (Scheuring, 
1928).  Trout  spermatozoa  have  been  a  favourite  object  for  investi- 
gations on  the  action  of  various  cations  and  anions  on  sperm  moti- 
lity (Scheuring,  1928;  Gaschott,  1928;  Schlenk,  1933;  Schlenk  and 
Kahmann,  1938).  Among  the  many  facts  brought  to  light  by  these 
investigations,  the  effect  of  potassium  ions  merits  particular  atten- 
tion. In  contrast  to  sodium  chloride,  a  diluent  containing  01 5% 
potassium  chloride  was  found  to  have  no  activating  effect  on  sperm 
motility,  and  moreover,  the  addition  of  potassium  ions  to  a  suspen- 
sion of  motile  sperm  in  sodium  chloride,  rendered  them  motionless. 
This  inhibition  by  potassium  ions,  however,  was  shown  to  be 
reversible  since  even  after  prolonged  storage  of  sperm  in  the  pre- 
sence of  potassium  ions,  it  was  still  possible  to  restore  their  motility 
by  dilution  with  water  or  sodium  chloride  solutions.  The  fact  that 
trout  spermatozoa  show  great  activity  upon  dilution  with  water  or 
sodium  chloride  solution  but  not  with  trout  seminal  plasma,  has 
been  attributed  by  Schlenk  to  the  high  potassium  content  of  the 
latter  (80  mg./lOO  ml.);  in  his  view,  the  rapid  increase  of  motility 
after  dilution  with  water  should  be  ascribed  to  the  decrease  of  potas- 
sium concentration  in  the  seminal  plasma  and  the  passage  of  potas- 
sium ions  from  the  sperm  cells  into  the  surrounding  medium. 
According  to  this  interpretation,  potassium  ions  fulfil  a  double 
function  in  semen:  they  preserve  the  sperm  energy  by  inducmg 
quiescence,  but  at  the  same  time,  they  engender,  as  it  were,  a  state 
of  preparedness  ('Bewegungsbereitschaft'). 

Much  information  on  the  effect  of  dilution  has  been  gathered 
from  experiments  with  sea-urchin  semen.  The  spermatozoa  of  sea- 
urchins,  unlike  those  of  man  and  higher  animals,  are  immotile  in 
the  absence  of  oxygen  (Harvey,  1930;  Barron,  1932).  In  sea-urchin 
semen  which  generally  has  a  high  sperm  density  (there  are  some 
2x10^°  cells /ml.  in  Echinus  esculentus),  the  spermatozoa  are 
motionless  so  long  as  they  remain  undiluted,  but  when  shed  into 
or  artificially  diluted  with,  sea-water,  they  become  intensely  motile 
and  with  increasing  dilution  their  oxygen  uptake  rises  as  well  (Gray, 
1928,  1931).  The  lack  of  sperm  movement  in  undiluted  sea-urchin 
semen  has  been  regarded  by  Gray  as  the  outcome  of  mechanical 
overcrowding;  each  cell  exercising  a  restraining  or  allelostatic  effect 
on  the  activity  of  its  neighbours.  Other  authors  believed  the  lack 

77?^  Influence  of  Extraneous  Factors  75 

of  movement  in  undiluted  semen  to  be  due  not  so  much  to  mutual 
restraint,  as  to  the  presence  of  a  specific  sperm-immobilizing  sub- 
stance either  in  the  sperm  or  in  the  seminal  plasma.  A  suggestion  has 
been  put  forward  that  the  main  cause  for  the  lack  of  motility  is  due 
to  the  sperm-paralysing  influence  of  androgamone  I.  Another  line 
of  thought  was  that  sperm  quiescence  is  due  to  the  seminal  plasma 
acting  by  virtue  of  its  high  potassium  content  or,  alternately,  its 
low  hydrogen  ion  concentration. 

The  various  theories  concerned  with  the  lack  of  sperm  movement 
in  undiluted  semen  have  been  reviewed  and  analysed  by  Rothschild 
(1948c,  1950^,  \95\a,b).  There  is,  he  argues,  no  conclusive  evidence 
that  the  spermatozoa  remain  motionless  in  undiluted  sea-urchin 
semen  because  of  any  one  factor  such  as  allelostasis,  sperm-immo- 
bilization, pH,  carbon  dioxide  or  potassium  ions.  It  is  particularly 
difficult,  he  points  out,  to  reconcile  the  observations  on  the  immo- 
bihzing  action  of  seminal  plasma  with  the  fact  that  seminal  plasma 
obtained  by  gentle  centrifugation  and  used  as  a  diluent  for  fresh 
semen,  renders  the  spermatozoa  as  active  as  sea-water.  He  found 
that  the  seminal  plasma  of  Echinus  esculentiis  acquires  sperm- 
immobilizing  properties  only  after  prolonged  centrifugation  of 
semen,  presumably  as  the  result  of  leakage  of  an  inhibitory  sub- 
stance from  the  cells;  the  rate  at  which  the  immobilizing  substance 
diffuses  from  the  spermatozoa  into  the  seminal  plasma  seems  to  vary 
and  this  probably  explains  the  conflicting  reports  concerning  the 
effect  of  centrifuged  seminal  plasma  on  sperm  motility.  Rothschild's 
own  experiments  indicate  that  the  main  cause  for  the  lack  of  sperm 
movement  in  undiluted  sea-urchin  semen  is  deficiency  of  oxygen; 
in  fact,  spermatozoa  can  be  mobilized  even  in  undiluted  semen  by 
increased  oxygen  tension,  and  deprived  of  motility  through  oxygen 
withdrawal.  According  to  this  author,  the  sudden  outburst  of 
activity  upon  dilution  of  semen,  is  simply  due  to  an  improved 
access  of  the  spermatozoa  to  oxygen. 

As  in  frog  and  fish,  the  marked  motility  evoked  in  sea-urchin 
sperm  by  dilution  is  only  of  limited  duration  and  is  subject  to  pro- 
gressive decline  in  spite  of  the  presence  of  oxygen.  If  the  dilution 
with  sea-water  is  very  excessive,  the  decline  may  almost  immediately 
follow  the  onset  of  activity.  Gray  (1928,  1931)  thought  that  the 
progressive  loss  of  activity  in  dilute  sperm  suspensions  could  be 

76  The  Biochemistry  of  Semen 

explained  by  spontaneous  and  irreversible  senile  decay  due  to  the 
gradual  disruption  of  cellular  organization,  exhaustion  of  food 
reserve,  depletion  of  energy,  and  autointoxication  with  the  reaction 
products  accumulated  during  the  period  of  activity.  He  showed  that 
it  is  possible  to  delay  the  decline  of  activity  by  replacing  the  sea- 
water  with  egg-water;  a  similar  effect  has  been  later  demonstrated 
with  solutions  of  the  egg-surrounding  jelly.  In  this  respect,  however, 
egg-water  cannot  be  looked  upon  as  a  very  specific  agent.  Both 
sperm  motility  and  respiration  can  be  extended,  for  instance,  by  the 
addition  of  seminal  plasma,  and  there  are  several  indications  that 
this  is  due  to  proteins  and  their  breakdown  products  in  the  seminal 
plasma.  Hayashi  (1945,  1946)  experimenting  with  Arbacia  punctu- 
lata,  demonstrated  the  occurrence  in  the  seminal  plasma  of  a  non- 
dialysable  constituent  beneficial  to  the  viability  and  fertilizing 
capacity  of  spermatozoa.  Wicklund  (1949,  1952)  demonstrated  a 
favourable  influence  of  serum  albumin  on  the  fertiUzing  capacity  of 
sea-urchin  spermatozoa;  she  found  that  the  fertilizing  power  of 
washed  or  aged  sperm  of  Psammechinus  miliaris  was  retained  much 
longer  following  dilution  with  albumin  solutions  than  with  sea- 
water.  Tyler  (1950)  and  Tyler  and  Atkinson  (1950)  found  that  the 
life-span  of  sea-urchin  sperm  can  be  considerably  extended  by  the 
addition  of  certain  peptides  and  amino  acids.  Tyler  and  Rothschild 
(1951)  examined  the  sperm  metabolism  of  Arbacia  punctulata  and 
Lytechinus  in  sea-water  enriched  with  amino  acids  and  noted  that 
under  such  experimental  conditions  the  initial  increase  of  respira- 
tion characteristic  of  the  'dilution  effect',  was  less  pronounced  but 
the  subsequent  decline  in  activity  .was  considerably  delayed,  and  the 
total  amount  of  oxygen  consumed  greatly  increased.  These  facts, 
coupled  with  evidence  of  non-utilization  of  the  added  amino  acids, 
indicated  that  the  amino  acids  enabled  the  spermatozoa  to  make 
fuller  use  of  their  endogenous  substrate,  probably  by  inducing  the 
formation  of  complexes  with  copper  and  other  toxic  heavy  metals 
commonly  present  in  sea-water.  This  hypothesis  has  gained  addi- 
tional support  from  the  results  of  further  work  on  the  detoxicating 
effect  of  metal-chelating  agents  such  as  ethylenediamine  tetra- 
acetate (versene),  diethyldithiocarbamate,  a-benzoinoxime  (cupron) 
and  8-hydroxyquinoline  (Tyler,  1953).  Perhaps  the  beneficial  action 
of  proteins  (Metz,  1945;  Wicklund,  1949)  and  of  seminal  plasma 

The  Influence  of  Extraneous  Factors  11 

(Hayashi,  1945,  1946;  Chang,  1949)  on  spermatozoa  is  also,  partly 
at  any  rate,  due  to  similar  processes. 

The  mechanism  of  inactivation  and  senescence  induced  in  sea- 
urchin  spermatozoa  by  prolonged  dilution  remains  obscure  but  there 
is  no  reason  to  suppose  that  it  differs  intrinsically  from  senescence 
in  mammalian  spermatozoa.  In  the  latter,  senescence  is  known  to  be 
associated  with  certain  definite  chemical  and  physical  changes,  such 
as  oxidation  of  intracellular  sulphydryl  groups  which  are  essential 
for  normal  motiUty,  decrease  in  the  content  of  adenosine  triphos- 
phate, and  increased  sperm  permeability,  which  leads  to  the  extra- 
cellular appearance  of  intracellular  sperm-proteins;  it  is  also  probable 
that  an  early  change  in  senescence  causes  swelHng  or  some  other 
degeneration  in  the  lipoprotein-containing  'lipid  capsule'  which 
normally  surrounds  the  sperm  cell  (see  p.  126).  It  is  by  no  means 
unlikely  that  some  upset  in  the  progress  of  ionic  exchange  reactions 
involving  particularly  potassium  ions,  is  also  linked  with  senescence. 

The  effect  of  dilution  on  mammalian  spermatozoa  is  essentially 
the  same  as  in  the  sperm  of  lower  animals.  Dilution  with  small 
volumes  of  saline  solution  produces  'activation'  or  excitation,  a 
phenomenon  well  known  to  Koelliker  and  other  investigators  of 
the  XlXth  century.  The  extent  of  this  activation  depends  of  course, 
on  concomitant  factors  such  as  pH,  temperature,  oxygen,  and  the 
presence  of  certain  substances.  For  example,  to  obtain  optimal 
motility  and  metabolism  in  diluted  bull  sperm.  Lardy  and  Phillips 
(1943^)  advise  the  addition  of  at  least  0'005m  potassium  and  0-012m 
magnesium,  with  simultaneous  omission  of  calcium  ions.  The  need 
for  potassium  ions  has  been  re-emphasized  by  Blackshaw  (1953). 
The  inclusion  of  phosphate,  mainly  as  a  buffer,  has  been  advocated 
repeatedly  by  several  authors,  even  though  in  higher  concentrations 
it  depresses  motility  and  respiration  of  bull  sperm  (Bishop  and 
Salisbury,  1954).  Sulphate  ions  have  been  recommended  by  Milo- 
vanov  on  the  ground  that  they  prevent  the  swelling  of  spermatozoa 
and  protect  the  'lipid  capsule'  from  the  action  of  sodium  chloride. 
In  the  writer's  laboratory,  the  following  salt  solution,  similar 
in  composition  to  the  Krebs-Henseleit-Ringer  solution,  is  used 
routinely  as  a  diluent  for  ram  and  bull  spermatozoa,  100  ml. 
0-9%  NaCl,  4  ml.  1-15%  KCl,  1  ml.  2-11%  KH2PO4,  1  ml.  3-82% 
MgSO.-VHaO,  2  ml.  of  a  1-3%  solution  of  NaHCOg  saturated  with 

78  The  Biochemistry  of  Semen 

CO2.  In  some  experiments  this  is  supplemented  with  (i)  0-5  g. 
fructose/ 100  ml.  ('Ringer-fructose'),  (ii)  20  ml.  0-25  M-phosphate 
buffer  pH  7-4,  the  latter  made  up  by  mixing  19  ml.  of  an  aqueous 
solution  containing  0-71  g.  Na2HP04  (or  1-79  g.  NasHPO^.UHaO) 
with  1  ml.  N-HCl  ('Ringer-phosphate')  or  (iii)  both  fructose  and 
phosphate  ('Ringer-fructose-phosphate').  Glass-distilled  water  and 
analytical  grade  reagents  are  used  throughout  (Mann,  1946^). 

If  the  dilution  of  mammalian  sperm  with  saline  is  excessive  it 
leads  after  a  short  spell  of  increased  activity  to  a  permanent  loss 
of  motility,  metabolic  activity  and  fertilizing  capacity.  According 
to  Milovanov  (1934),  the  resistance  (R)  of  sperm  to  the  immobiliz- 
ing effect  of  dilution  with  large  volumes  of  1%  NaCl,  varies  in 
different  animals;  he  believes  that  specimens  of  semen  capable  of 
high  resistance  possess  at  the  same  time  high  fertilizing  capacity. 

The  formula  R=—  used  by  Milovanov  in  the  'resistance  test',  is  cal- 


culated  from  the  volume  of  1%  NaCl  (V)  required  to  arrest  in  a 
given  volume  of  semen  (v)  all  progressive  movement  of  sperm-heads. 
In  bull  semen  the  test  is  carried  out  with  002  ml.  semen,  at  17-24°, 
and  sperm  motility  is  assessed  by  microscopic  examination  after  the 
addition  of  successive  lots  of  10  ml.  1%  NaCl  solution  (Nagornyi 
and  Smimov,  1939).  The  R  values  given  by  Milovanov  are:  bull 
300-20,000,  ram  100-5000,  stallion  100-1500,  boar  60-1000,  dog 
200-600.  Some  doubts  however,  have  arisen  as  to  the  significance  of 
Milovanov's  test  in  the  assessment  of  male  fertility,  as  well  as  his 
statement  that  the  immobilizing  action  is  due  to  the  toxicity  of 
sodium  chloride  as  such  (Emmens  and  Swyer,  1947,  1948;  Cheng, 
Casida  and  Barrett,  1949).  But  there  is  general  agreement  that 
excessive  dilution  is  invariably  harmful  to  spermatozoa.  This  is 
reflected,  among  others,  in  Chang's  (1946)  finding  that  a  constant 
number  of  rabbit  spermatozoa  has  a  greater  fertilizing  capacity 
in  a  small,  as  opposed  to  a  large,  volume  of  salt  diluent.  Chang  insem- 
inated superovulated  does  with  saline  suspensions  of  spermatozoa, 
removed  the  ova  38^2  hr.  later,  and  counted  the  cleaved  ova. 
Results  showed  that  the  insemination  of  a  total  of  30-40  thousand 
spermatozoa  in  1  ml.  0-9^o  NaCl  was  followed  by  the  cleavage  of 
0-6%  of  ova,  whereas  the  same  number  of  cells  suspended  in  0- 1  ml. 
produced  cleavage  in  17-42%  of  ova.  Another  observation  which 

The  Influence  of  Extraneous  Factors  79 

confirms  the  adverse  effect  of  dilution  is,  that  although  the  addition 
of  fructose  to  an  isotonic  salt  diluent  prolongs  the  metabolic  activity 
of  bull  sperm  suspensions,  excessive  dilution  with  'Ringer-fructose' 
solution  leads  to  a  decline  in  the  rate  of  fructolysis  (Mann  and 
Lutwak-Mann,  1948).  Up  to  a  point,  the  changes  due  to  the  'dilution 
effect'  resemble  those  produced  by  extensive  washing  of  spermatozoa. 
Thus  for  example,  whereas  one  careful  washing  of  centrifuged  ram 
sperm  with  several  volumes  of  Ringer  solution  causes  but  negligible 
damage,  repeated  washing  results  in  a  progressive  decline  in  motility 
and  metabolism  (Mann,  1945^;  White,  1953). 

The  use  of  artificial  diluents  in  the  storage  of  semen 

It  has  been  noticed  repeatedly  that  the  deleterious  effect  of  salt 
diluents  on  spermatozoa  can  be  at  least  partly  overcome  by  the 
inclusion  of  certain  organic  substances,  mainly  of  colloidal  nature. 
These  observations  stimulated  the  development  of  the  so-called 
'media',  'pabula',  'dilutors'  and  'extenders',  for  use  in  the  storage 
of  semen  for  artificial  insemination. 

The  use  of  diluents  in  the  storage  of  semen  has  its  origin  in 
certain  experiments  made  by  Donne,  de  Quatrefages,  and  Koelliker, 
who  examined  the  effect  of  blood  plasma,  milk,  various  proteins 
and  sugars,  on  the  spermatozoa.  Early  in  this  century,  Hirokawa 
(1909),  Champy  (1913),  and  others,  experimented  with  blood  plasma 
and  serum.  Particularly  illuminating  results  were  obtained  by 
Grodzinski  and  Marchlewski  (1938)  who  stored  cock  semen  diluted 
ten  times  with  chicken  serum  at  2°  for  periods  up  to  eight  days, 
and  found,  on  increasing  the  temperature  to  37°,  that  the  sperma- 
tozoa were  motile.  In  addition,  however,  these  authors  also  found, 
in  agreement  with  Bernstein  and  Lazarev  (1933),  that  it  is  advisable 
to  use  blood  serum  which  has  been  pre-treated  at  55°,  as  otherwise 
the  spermatozoa  tend  to  agglutinate.  An  agglutinating  and  spermi- 
cidal factor,  present  in  fresh  serum  but  inactivated  by  a  few  days' 
storage  at  4°,  or  10  minutes'  heating  at  56°,  was  found  by  Chang 
(1947a)  in  human,  bovine,  rabbit,  guinea  pig,  and  rat  serum.  It  is 
mainly  because  of  its  agglutinating  properties  that  blood  serum  has 
not  found  a  wider  application  as  a  semen  diluent.  On  the  other 
hand,  as  a  direct  result  of  immunological  studies  on  the  reactions 
between  blood  serum  and  spermatozoa,  rapid  progress  has  been 

80  The  Biochemistry  of  Semen 

made  in  studies  concerning  spermatozoal  antigens  and  antibodies, 
culminating  in  the  development  of  several  antispermatozoal  sera, 
active  not  only  tov^ards  heterologous  but  also  homologous,  sperma- 
tozoa. So  far,  most  attempts  to  immunize  a  female  against  sperma- 
tozoa of  her  own  species,  as  a  means  of  'serological  contraception', 
have  been  failures.  A  wealth  of  data  on  sperm  immunology  and 
sperm-serum  interaction  can  be  found  in  articles  by  Metchnikoff 
(1900),  Moxter  (1900),  Godlewski  (1912),  Kato  (1936),  Henle  and 
Henle  (1940),  Parkes  (1944),  Snell  (1944),  Tyler  (1948),  Smith  (1949), 
Docton  et  al.  (1952),  and  Kibrick  et  al.  (1952). 

One  of  the  first  diluents  used  in  veterinary  practice  for  the  in- 
semination of  cattle  in  Russia,  was  Milovanov's  'SGC-2  dilutor' 
(13-6  g.  Na2S04,  12  g.  glucose,  and  5  g.  Witte's  peptone,  in  1  1. 
water).  The  same  investigator  experimented  also  with  so-called 
gelatinized  diluents  which  contained  apart  from  salts  and  glucose, 
some  gelatin,  so  as  to  endow  the  fluid  with  a  jelly-like  consistency. 
One  such  diluent  developed  for  the  storage  of  bull  semen,  has  been 
the  'GPC-3-G  dilutor'  (1-7  g.  Na2HP04,  007g.  KH0PO4,  008  g. 
Na2S04,  2-85  g.  glucose,  5  00  g.  gelatin,  1  1.  water.)  A  method 
adopted  by  the  Russian  workers  was  to  use  the  diluted  semen  for 
insemination  in  the  form  of  gelatin  capsules.  A  major  advance  in 
the  technique  of  semen  storage  has  been  the  introduction  by 
Phillips  and  Lardy  (1939,  1940)  of  the  'egg-yolk-phosphate  diluent' 
which  became  widely  established  both  in  America  and  in  Europe 
for  the  purpose  of  preservation,  transportation  and  insemination  of 
bull  semen.  It  is  prepared  by  mixing  freshly  separated  egg  yolk  with 
an  equal  volume  of  phosphate  buffer,  pH  7-4  (2  g.  NagHPOi-  OHgO^ 
0-2  g.  KH2PO4,  made  up  to  100  ml.  with  water).  For  actual  storage,  | 
and  subsequent  insemination,  bull  semen  is  diluted  up  to  100  times, 
or  more,  with  the  egg-yolk  phosphate  dilutor.  In  addition  to  pre- 
serving sperm  viability,  the  dilutor  protects  the  spermatozoa  effi- 
ciently from  'temperature  shock',  that  is  from  the  rapid  immo- 
bilization induced  by  sudden  cooling  of  semen  to  5-10°,  the  usual 
temperature  for  storage  of  semen  (Chang  and  Walton,  1940;  Easley, 
Mayer  and  Bogart,  1942).  The  chemical  nature  of  the  protecting 
substance  is  unknown  but  there  are  indications  that  it  is  an  acetone- 
soluble  but  ether-insoluble,  compound  (Mayer  and  Lasley,  1944). 

Many  recommendations  have  been  made  to  improve  the  egg-yolk- 

The  Influence  of  Extraneous  Factors  81 

buffer  diluent  by  the  inclusion  of  various  additives  such  as  gela- 
tine (Knoop,  1941),  glycine  (Knoop  and  Krauss,  1944;  Tyler  and 
Tanabe,  1952),  sodium  citrate  (Willett  and  Salisbury,  1942;  Salis- 
bury, Knodt  and  Bratton,  1948),  bicarbonate  and  glucose  (Kamp- 
schmidt,  Mayer,  Herman  and  Dickerson,  1951),  dialysed  yolk 
(Tosic  and  Walton,  \9A6a)  and  liquid  whole  egg  (Dunn  and  Bratton, 
1950);  to  counteract  the  danger  from  bacterial  contamination,  cer- 
tain antibiotics  are  sometimes  added  such  as  penicillin,  strepto- 
mycin, polymixin,  aureomycin  or  sulphonamide  drugs.  On  this  sub- 
ject alone  there  is  a  vast  number  of  publications  of  which  only  a 
few  can  be  quoted  here  (Knodt  and  Salisbury,  1946;  Bay  ley,  Cobbs 
and  Barrett,  1950;  Branton,  James,  Patrick  and  Newsom,  1951; 
Foote  and  Bratton,  1949,  1950;  Hennaux,  Dimitropoulos  and 
Cordiez,  1947;  Pursley  and  Herman,  1950;  VanDemark,  Salisbury 
and  Bratton,  1949;  VanDemark,  Bratton  and  Foote,  1950;  Willett, 
1950;  Dunn,  Bratton  and  Henderson,  1953). 

Some  attempts  were  made  a  while  ago  to  replace  the  egg-yolk 
buffer  diluent  by  a  chemically  more  clearly  defined,  artificial 
medium.  Thus,  Phillips  and  Spitzer  (1946)  developed  the  so-called 
*L.G.B.-pabulum'  which  contained  as  essential  ingredients  1-2%  of 
a  lipid  fraction  (L),  made  up  of  lipositol  (an  inositol-containing 
phospholipid),  0-6%  glucose  (G),  phosphate  buffer  (B),  pH  7-4 
(0-2%  KH2PO4  and  2%  Na2HP04-12H20),  with  0-2%  galactose, 
003%  of  sulphasuxidine  or  streptomycin,  and  lastly  3%  gum  acacia, 
added  to  provide  'sufficient  body  to  prevent  the  settling  out  of  sperm 
upon  standing  in  storage'.  Other  substitutes  for  egg-yolk  which 
have  been  suggested  at  various  times,  include  milk,  glycerol,  paraffin, 
arachis  oil  and  synthetic  plasma-substitutes  such  as  'periston' 
(Laplaud,  Bruneel  and  Galland,  1951;  Koch  and  Robillard,  1945; 
Rostand,  1946,  1952;  Asher  and  Kaemmerer,  1950;  Thacker  and 
Almquist,  1953). 

It  remains  for  future  investigations  to  invent  an  ideal  diluent. 
Such  a  diluent  would  be  expected  to  combine  the  following  features, 
isotonicity,  efficient  buffering  capacity,  nutrient  value,  antibacterial 
potency,  stabilizing  action  of  a  'protective  colloid',  anti-oxidant 
ability,  and  above  all,  good  keeping  quality  in  a  ready-to-use  form. 
Furthermore,  it  should  protect  semen  from  the  effects  of  sudden 
changes  of  temperature  and  preserve  its  full  fertilizing  capacity  for  a 

82  The  Biochemistry  of  Semen 

reasonable  period  at  the  low  temperatures  of  storage  in  vitro.  There 
is  also  the  problem  as  to  whether  semen  should  be  stored  in  a 
diluted  form  or  whether  it  would  be  better  to  dilute  it  just  before 
actual  use.  It  would  appear  that  on  the  whole,  under  natural  in  vivo 
conditions,  spermatozoa  survive  best  in  a  highly  concentrated  state 
when  their  motility  is  reduced  to  a  minimum;  the  prolonged  life-span 
of  sperm  in  the  epididymis  certainly  points  in  that  direction.  Another 
even  more  suggestive  example  is  the  behaviour  of  ejaculated  bat 
sperm:  the  density  of  sperm  as  found  in  the  bat  uterus  after  copula- 
tion is  very  high,  about  6  million  cells //^l.  (Schwab,  1952);  in  this 
condition  the  spermatozoa  seem  to  be  largely  immotile,  but  are 
nevertheless  capable  of  survival  for  several  months.  Such  spermato- 
zoa respond  to  artificial  dilution  by  becoming  intensely  motile,  but 
then  they  survive  for  not  longer  than  a  few  days. 


Intracellular  Enzymes,  Metalloproteins, 

Nucleoproteins,  and  other  Protein  Constituents 

of  Spermatozoa 

Mechanical  separation  of  sperm  from  seminal  plasma;  release  of  intra- 
cellular proteins  from  damaged  spermatozoa.  Removal  of  the  sperm 
nucleus  from  the  cytoplasm.  Protein-bound  iron,  zinc,  and  copper.  Cyto- 
chrome. Catalase.  Hyaluronidase  and  other  'lytic'  agents.  Sperm  nucleo- 
proteins. Deoxyribonucleic  acid.  The  basic  nuclear  proteins;  protamines 
and  histones.  The  non-basic  nuclear  proteins;  karyogen  and  chromosomin. 
Keratin-like  protein  of  the  sperm  membrane. 

Mechanical  separation  of  sperm  from  seminal  plasma;  release  of  intra- 
cellular proteins  from  damaged  spermatozoa 
A  STUDY  of  the  proteins  present  in  the  spermatozoa  themselves  calls 
for  an  efficient  separation  of  the  sperm  from  the  seminal  plasma,  by 
centrifugation  and  washing.  However,  the  spermatozoa  are  filiform 
structures,  highly  vulnerable  to  mechanical  damage.  Centrifugation, 
dilution  and  washing  may  inflict  an  injury  upon  the  sperm  cell  which, 
even  if  not  apparent  upon  ordinary  microscopic  examination,  never- 
theless results  in  a  leakage  of  certain  proteins  from  the  spermatozoa 
into  the  surrounding  medium.  Thus,  for  instance,  cytochrome  c  is 
easily  detached  from  the  sperm  structure  as  a  result  of  cellular 
damage  or  prolonged  storage  of  spermatozoa  (Mann,  1951a);  be- 
cause of  that,  the  spectroscopic  detection  of  extracellular  cyto- 
chrome c  provides  a  sensitive  indicator  of  'senescence'  changes  in 
spermatozoa.  Another  example  is  the  release  of  hyaluronidase  by 
the  spermatozoa  (see  p.  94).  At  one  time,  this  phenomenon  was 
ascribed  to  a  true  secretory  function  of  the  normal  sperm  cells 
but  more  recent  evidence  suggests  that  the  liberation  of  hyaluroni- 
dase takes  place  in  an  ageing  or  moribund  cell  population.  Yet 
another  phenomenon  in  this  category  is  the  loss  of  lipoprotein 
from  the  'lipid  capsule'  of  the  sperm  cell  which  may  easily  occur  as 
7  83 

84  The  Biochemistry  of  Semen 

a  result  of  extensive  washing.  However,  even  if  the  separation  of 
sperm  from  the  seminal  plasma  has  been  carried  out  with  due  care 
and  attention,  there  is  no  certainty  that  a  loss  of  intracellular  pro- 
tein has  not  been  incurred.  For  this  reason,  one  cannot  but  view 
with  suspicion  the  results  of  protein  analyses  in  sperm,  if  they  have 
been  performed  with  spermatozoa  centrifuged  at  high  speed,  or 
washed  extensively  with  large  volumes  of  diluents,  some  of  them 
anisotonic  or  unbuffered. 

The  data  at  present  available  indicate  that  spermatozoa  have 
a  much  higher  concentration  of  proteins  than  the  seminal  plasma. 
Friedrich  Miescher  (1870,  1878,  1897)  whose  fundamental  studies 
provided  the  earliest  information  on  the  chemical  nature  of  some 
of  the  sperm  proteins,  was  also  the  first  to  point  out  that  in  salmon, 
for  instance,  the  high  dry  weight  and  protein  content  of  semen  was 
almost  entirely  due  to  the  spermatozoa,  whereas  the  seminal  plasma 
gave  practically  no  precipitate  with  2  vol.  of  acidified  ethanol,  and 
contained  no  more  than  0-78%  dry  matter,  of  this  0-65%  mineral, 
and  only  013%  organic,  material.  However,  a  more  recent  analysis 
of  Sabno  fontinalis  has  shown  a  content  of  1  -76%  nitrogen  and 
0-43%  phosphorus  in  the  seminal  plasma  (Felix,  Fischer,  Krekels 
and  Mohr,  1951).  Sea-urchin  {Arbacla  punctulata)  seminal  plasma 
has  about  0-25%  protein  (Hayashi,  1945). 

Table  10.  Protein  composition  of  bull  semen 
(Sarkar  et  al,  1947) 

In  dried  material,  ash  and  lipid-free 


Seminal  plasma 

Total  nitrogen 





















Glutamic  acid 



Protein  Constituents  of  Spermatozoa  85 

On  the  mammalian  side,  bull  semen  has  received  much  attention 
from  protein  analysts.  Zittle  and  O'Dell  (1941«,  6)  investigated  the 
nature  of  the  sulphur  in  bull  sperm  and  found  that  over  two-thirds  of 
the  1-6%  S  present  in  lipid-free  dry  material  is  accounted  for  by 
cystine  and  cysteine,  and  the  remainder  by  methionine.  Sarkar, 
Luecke  and  Duncan  (1947)  whose  results  are  shown  in  Table  10, 
analysed  separately  frozen-dried  bull  spermatozoa  (20  g.  dry  material 
from  100  g.  fresh  washed  sperm)  and  seminal  plasma  (1-4  g.  dry 
material  from  100  ml.)  for  total  nitrogen  and  amino  acid  content. 
The  amino  acids  were  assayed  by  microbiological  methods  in  protein 
hydrolysates;  however,  with  the  exception  of  arginine,  and  to  a  small 
extent  leucine  and  tryptophan,  the  result  of  the  amino  acid  analysis 
failed  to  reflect  the  different  physical  character  and  physiological 
function  of  proteins  in  spermatozoa  and  seminal  plasma.  The  con- 
spicuously high  content  of  arginine  in  the  spermatozoa  is,  of  course, 
due  to  the  presence  of  this  amino  acid  in  the  nucleoprotein,  but  even 
in  the  seminal  plasma  the  proportion  of  arginine  exceeds  consider- 
ably that  of  any  other  amino  acid,  with  the  possible  exception  of 
glutamic  acid.  Further  data  on  the  composition  of  bull  sperm  pro- 
tein have  been  presented  by  Porter,  Shankman  and  Melampy  (1951) 
who  found  in  extensively  washed,  lipid-free  and  dried  spermatozoa 
16-7%  nitrogen;  m  addition  to  the  amino  acids  recorded  previously, 
they  identified  aspartic  acid  (5%),  glycine  (1-7%),  proHne  (3-1%), 
serine  (4-5%)  and  tyrosine  (4-3%). 

There  is  but  little  information  apart  from  some  immunological 
studies,  on  the  chemical  differences  between  the  sperm  proteins  of 
various  species.  An  early  attempt  in  this  direction  was  made  by 
Faure-Fremiet  (1913)  who  purified  'ascaridine',  a  protein  peculiar 
to  the  testicular  tissue,  and  probably  also  to  spermatozoa,  of 
Ascaris  megalocephala;  an  interesing  account  of  this  and  other 
unusual  characteristics  of  Ascaris  sperm  is  given  by  Panijel  (1951). 

Removal  of  the  sperm  mwleus  from  the  cytoplasm 

Special  techniques  are  required  to  sever  the  sperm-head  from  the 
tail,  as  a  preliminary  to  protein  analysis  in  these  two  morphological 
components  of  the  sperm  cell.  Miescher,  who  pioneered  in  this  field, 
selected  for  his  studies  fish  spermatozoa  where  a  separation  can  be 
accomplished  relatively  easily  with  water  or  dilute  organic  acids, 

86  The  Biochemistry  of  Semen 

which  plasmolyse  the  tails  (together  with  the  middle-pieces),  but  not 
the  heads.  In  this  way  he  obtained  by  centrifugation  two  portions,  a 
supernatant  fluid  representing  the  cytoplasm,  and  a  deposit  con- 
sisting of  sperm-heads  which  could  be  further  purified  by  washing 
with  water. 

According  to  Miescher's  calculations,  in  salmon  spermatozoa  the 
heads  and  tails  contribute  76  and  24%  of  fresh  material,  and  87  and 
13%  of  the  lipid-free  material,  respectively.  Suspensions  of  fish 
sperm-heads  obtained  by  plasmolysis,  centrifugation  and  washing, 
are  largely  sperm  nuclei  and  that  is  why  they  have  been  used  exten- 
sively for  the  study  of  nucleoproteins  by  Miescher  and  others  who 
followed  in  his  footsteps.  It  is,  however,  rather  uncertain  what  pro- 
portion of  cytoplasm  defies  aqueous  extraction  and  how  much 
protein  is  lost  from  the  sperm-heads  in  the  course  of  washing.  It  is 
quite  likely  that  losses  of  varying  magnitude  occur,  which  would 
account  for  the  discrepancies  in  analytical  results  obtained  by 
different  authors,  particularly  as  regards  the  content  of  cytoplasmic 
and  non-basic  nuclear  proteins  of  fish  spermatozoa. 

The  supernatant  fluid  obtained  by  centrifugation  of  plasmolysed 
salmon  spermatozoa  was  found  by  Miescher  to  be  rich  in  soluble 
proteins  and  lipids.  On  addition  of  ethanol  he  obtained  two  frac- 
tions, one  which  was  ethanol-insoluble,  accounted  for  41-9%  of  dry 
material  and  contained  mainly  protein  (C  51-85,  H  7-10,  N  14-94, 
S  1-37),  and  the  other  ethanol-soluble,  equal  to  51-8%  of  dry 
material  and  made  up  of  lecithin,  fat  and  cholesterol.  Salmon 
sperm  cytoplasm  is  known  to  contain  phosphatases  active,  amongst 
others,  towards  adenosine  triphosphate;  it  is  devoid  of  deoxynucleo- 
proteins  but  contains  apparently  some  ribonucleic  acid  and  several 
free  amino  acids,  namely  alanine,  valine,  isoleucine,  tyrosine,  aspar- 
tic  acid,  and  glutamic  acid  (Felix  et  al.,  1951). 

Mammalian  spermatozoa,  in  contrast  to  those  of  fishes,  cannot  be 
plasmolysed,  and  their  heads  do  not  come  off"  in  water  or  acid.  To 
overcome  this  obstacle,  Zittle  and  O'Dell  (\94la,  b)  exposed  bovine 
epididymal  spermatozoa  to  ultrasonic  waves  and  in  this  way  dis- 
sociated the  sperm-heads  from  the  middle-pieces  and  tails.  On  slow 
centrifugation  of  the  disintegrated  sperm  suspensions,  the  heads 
settled  out  first;  at  increasing  speed,  the  middle-pieces  also  formed 
a  sediment,  leaving  in  the  supernatant  fluid  most  of  the  fragmented 

Protein  Constituents  of  Spermatozoa 


tails.  The  products  thus  obtained  were  extracted  with  lipid  solvents 
and  dried.  The  lipid-free  dry  weights  of  heads,  middle-pieces  and 
tails  were  51,  16,  and  33%,  respectively,  of  the  whole  spermatozoa. 
The  content  of  ash,  nitrogen,  phosphorus,  sulphur,  cystine,  and 
methionine  in  the  three  fractions  is  shown  in  Table  11. 

Table  11.  Composition  of  sperm-heads,  middle-pieces,  and  tails, 

dissociated  by  ultrasonic  disintegration  of  bull  spermatozoa 

(Zittle  and  O'Dell,  1941«) 

In  dried,  lipid-free  material  (%) 





Whole  sperm 
































*  Corresponding  to  a  content  of  40-5%  deoxyribonucleic  acid. 

In  the  author's  experience  (Mann,  1949,  19516),  a  relatively  simple 
procedure  for  the  disruption  of  ram  spermatozoa  is  to  shake  them 
with  fine  glass  beads  in  the  mechanical  disintegrator  of  Mickle 
(1948).  Such  treatment  leads  to  fragmentation  of  the  middle-pieces 
and  tails,  though  not  of  the  sperm-heads,  and  yields  on  slow  centri- 
fugation  a  yellow-coloured,  opalescent  fluid  which  probably  repre- 
sents the  sperm  cytoplasm.  This  material  is  very  rich  in  enzymes;  it 
contains  among  others,  the  intermediary  enzymes  of  fructolysis, 
certain  phosphatases,  and  the  complete  cytochrome-cytochrome 
oxidase  system,  as  well  as  a  potent  succinic  dehydrogenase,  the 
activity  of  which  can  be  demonstrated  both  by  methylene-blue  reduc- 
tion and  by  oxygen  uptake  in  presence  of  succinate.  The  succinic 
dehydrogenase  activity  shown  by  disintegrated  ram  spermatozoa 
contrasts  strikingly  with  the  behaviour  of  the  fresh  intact  sperm 
cells,  the  O2  consumption  of  which  is  not  markedly  enhanced  by  the 
addition  of  succinate.  The  difference  in  enzymic  behaviour  between 
the  intact  and  disrupted  sperm  cells  has  something  of  a  parallel  in 
the  activity  of  blood  carbonic  anhydrase  which  can  be  demonstrated 
much  more  readily  in  laked  than  in  unlaked,  erythrocytes  (Keilin 
and  Mann,  1941). 

88  The  Biochemistry  of  Semen 

The  heads  of  ram  spermatozoa  separated  by  the  process  of 
mechanical  disintegration  can  be  further  freed  from  hpoprotein 
and  from  adhering  particles  of  middle-pieces  and  tails  by  repeated 
washing  and  differential  centrifugation.  Preparations  obtained  in 
this  way  consist  of  sperm-heads  only;  they  were  found  to  contain 
3  •9-4-3%  phosphorus,  all  of  it  accounted  for  by  deoxyribonucleic  acid, 
but  were  free  from  lipid  and  acid-soluble  phosphorus  compounds. 

Protein-bound  iron,  zinc  and  copper 

Zittle  and  Zitin  (1942^)  found  that  the  total  iron  content  of  dried 
lipid-free  bovine  epididymal  sperm  is  about  7  mg./lOO  g.,  more  iron 
being  present  in  the  middle-pieces  and  tails  than  in  the  heads.  Of 
the  total  iron,  60%  was  extractable  with  pyrophosphate  and  tri- 
chloroacetic acid  at  100^,  and  was  therefore  assumed  to  be  of  non- 
haematin  nature;  an  attempt  to  identify  haematin  in  the  non- 
extractable  portion  was  unsuccessful.  However,  with  the  aid  of  a 
spectroscopic  method  (Mann,  1937,  1938)  designed  specifically  for 

Table  12.  Distribution  of  total  iron,  zinc  and  copper  in  ram  semen 
(Mann,  1945a) 


100  ml.  semen  contain 

In  sperm  (23  ml.; 
3-5x10"  cells) 

In  seminal 
plasma  (77  ml.) 

Fe  (mg.) 
Zn  (mg.) 
Cu  (mg.) 



determination  of  haematin  (as  pyridine  haemochromogen)  in  animal 
and  plant  tissues,  the  author  was  able  to  detect  readily  and  to  deter- 
mine quantitatively  haematin  in  bull  as  well  as  in  ram  spermatozoa. 
The  distribution  in  ram  semen,  of  total  iron,  and  also  of  zinc  and 
copper,  is  shown  in  Table  12  (Mann,  1945^).  It  can  be  seen  that  in 
the  ram  the  concentration  of  these  three  elements  is  much  higher 
in  the  spermatozoa  than  in  the  seminal  plasma.  With  the  aid  of  a 
Mickle  disintegrator,  it  was  possible  to  separate  the  heads  from  the 
tails  and  middle-pieces  and  to  obtain  sperm-head  preparations  which 
contained  some  iron  but  were  completely  free  from  haematin.  On 
the  other  hand,  the  'homogenates'  from  disintegrated  tails  and 

Protein  Constituents  of  Spermatozoa 


middle-pieces  contained  a  high  proportion  of  iron  in  the  form  of 
haematin,  as  can  be  seen  from  Table  13.  Iron  as  well  as  copper  and 
zinc,  present  in  the  tail  and  middle-piece  of  ram  spermatozoon,  is 
largely  non-dialysable.  Iron  occurs  mainly  as  haematin  some  of 
which  appears  to  be  free  and  the  rest  protein-bound,  mostly  in  the 
form  of  cytochrome.  Copper  belongs  to  a  protein  complex  which 
readily  gives  up  the  metal  on  treatment  with  acid,  thus  resembling 
haemocuprein,  the  copper-protein  isolated  some  time  ago  from 
blood  cells  (Mann  and  Keilin,  1938).  Zinc  also  forms  a  complex  with 
a  protein  but  unlike  the  zinc-protein  of  blood  cells  it  has  negligible 
carbonic  anhydrase  activity. 

Table  13.  Content  of  total  iron,  haematin,  zinc,  and  copper ^  in 
mechanically  disintegrated  middle-pieces  and  tails  of  ram  spermatozoa 

(Spermatozoa  separated  from  seminal  plasma  by  centrifugation  and 
washing;  mechanically  disrupted  in  Mickle's  disintegrator;  sperm-heads 
removed  by  centrifugation.  The  supernatant  fluid  ('homogenate')  which 
represents  disintegrated  middle-pieces  and  tails  was  analysed  before  and 
after  dialysis.  Its  dry  weight,  expressed  as  mg./lOO  ml.  semen,  was  3300 
before,  and  2960  after,  dialysis.) 

In  the  homogenate  from  mid-pieces  and  tails 

Total  contents 


(mg./lOO  ml. 

(mg./lOO  g. 

dr.  wt.  of 


(mg./lOO  ml. 

(mg./lOO  g. 

dr.  wt.  of 


Total  Fe 





Haematin  Fe 















Human  semen,  in  contrast  to  ram,  has  a  much  higher  zinc 
content.  The  first  to  take  notice  of  this  were  Bertrand  and  Vladesco 
(1921)  who  found  5-3-220  mg.  Zn/100  ml.  semen.  More  recently, 
the  problem  of  seminal  zinc  was  taken  up  by  Mawson  and  Fischer 
(1953)  who  found  that  apart  from  the  high  zinc  content  of  human 
seminal  plasma  which  is  derived  from  the  prostatic  secretion  (see 
p.  19),  centrifuged  spermatozoa  of  man  also  carry  a  considerable 
zinc  reserve  of  their  own,  nearly  2  mg./g.  dry  matter.  Of  this,  how- 
ever, only  a  minute  fraction  is  endowed  with  carbonic  anhydrase 

90  The  Biochemistry  of  Semen 

Sea-urchin  semen  has  approximately  the  same  concentration 
of  copper  as  ram  semen,  with  a  similar  distribution  of  the  metal 
between  sperm  and  seminal  plasma  (Barnes  and  Rothschild,  1950). 


Early  investigators  of  semen  were  well  aware  of  the  fact  that 
spermatozoa  give  a  positive  indophenol  reaction  with  the  'Nadi* 
reagent,  particularly  marked  in  the  regions  of  the  acrosome  and 
middle-piece  (Herwerden,  1913).  Ostwald  (1907),  Voss  (1922)  and 
Sereni  (1929),  among  others,  made  important  contributions  to  the 
subject  of  sperm  indophenol  oxidase  and  noted  that  the  intensity  of 
the  reaction  increased  towards  the  final  stages  of  sperm  maturation 
and  after  ejaculation. 

When  in  1925  Keilin  discovered  cytochrome  and  identified 
indophenol  oxidase  with  cytochrome  oxidase,  he  noticed  that  of  all 
the  organs  of  a  perfused  frog,  the  heart  muscle  and  the  testicular 
tissue  exhibited  the  strongest  absorption  spectrum  of  cytochrome. 
A  little  later,  Brachet  (1934)  reported  the  presence  of  cytochrome  in 
frog  sperm,  and  Ball  and  Meyerhof  (1940)  in  sea-urchin  spermatozoa. 
In  spite  of  that,  attempts  by  several  workers  to  detect  the  spectrum 
of  cytochrome  in  mammalian  spermatozoa  met  with  failure  and  the 
functioning  of  cytochrome  in  mammalian  semen  continued  to  be 
deduced  only  indirectly  from  the  evidence  based  on  the  oxidation 
of  succinate  and  phenylenediamine  (Shergin,  1940;  Lardy  and 
Phillips,  1941c;  Zittle  and  Zitin,  1942^;  MacLeod,  1943a).  This  led 
to  some  speculation,  particularly  in  the  case  of  human  semen,  about 
the  mechanism  of  respiration,  the  more  so,  since  it  has  been  asserted 
that  the  oxygen  consumption  of  human  semen  is  associated  pre- 
dominantly with  the  seminal  plasma  and  not  with  the  spermatozoa 
themselves  (MacLeod,  1941a;  Ross,  Miller  and  Kurzrok,  1941; 
Zeller,  1941). 

In  an  efTor^  to  re-examine  the  whole  problem,  the  author  made  a 
study  of  the  cytochrome  content  of  mammalian  spermatozoa  (Mann, 
1945a,  c).  With  the  aid  of  the  microspectroscope,  an  instrument 
eminently  suitable  for  direct  observation  of  absorption  bands  in 
tissues,  no  difficulty  was  experienced  in  the  detection  of  the  com- 
plete cytochrome  spectrum  in  both  ram  and  bull  semen.  Human 
semen  has  a  sperm  density  at  least  ten  times  lower  than  bull  semen, 

Protein  Constituents  of  Spermatozoa  91 

but  even  here,  the  demonstration  of  the  absorption  bands  of  cyto- 
chrome became  possible  (Mann,  \95\d)  by  the  application  of  the 
technique  of  Keilin  and  Hartree  (1949,  1950),  whereby  manifold 
intensification  of  absorption  bands  is  brought  about  by  means  of 
liquid  air. 

Our  evidence  for  the  occurrence  and  active  participation  of  the 
cytochrome  system  in  the  oxidative  metabolism  of  mammalian 
spermatozoa  can  be  briefly  summarized  as  follows.  Whole  fresh 
semen  examined  a  little  while  after  ejaculation  shows  the  diffuse 
spectrum  of  oxidized  cytochrome  as  well  as,  weakly,  the  absorption 
bands  of  reduced  cytochrome  a,  b,  and  c.  However,  after  the  addi- 
tion of  a  reducing  agent  or  on  anaerobic  incubation  of  the  semen, 
the  bands  of  the  reduced  cytochromes  become  much  more  pro- 
nounced, the  cytochrome  a  band  being  more  distinct  than  c,  and 
the  latter  stronger  than  cytochrome  b.  The  picture  is  similar  with 
washed  sperm  suspensions  in  fructose-Ringer-phosphate  solution; 
freshly  prepared  sperm  suspensions  show  mainly  the  spectrum  of 
oxidized  cytochrome,  which  becomes  reduced  in  the  course  of 
anaerobic  incubation;  on  aeration  of  the  incubated  suspension 
cytochrome  reverts  to  the  oxidized  form.  The  band  of  cytochrome  a 
can  be  shown  to  undergo  a  typical  change  under  the  influence  of 
carbon  monoxide;  the  reaction  product  thus  formed  in  the  sperm 
resembling  closely  the  carbon  monoxide  compound  of  cytochrome 
oxidase  or  cytochrome  a^,  originally  described  by  Keilin  and 
Hartree  (1939)  in  heart  muscle  preparations.  Carbon  monoxide, 
cyanide,  azide,  hydroxylamine,  and  other  typical  inhibitors  of  the 
cytochrome  system  in  respiring  tissues,  all  inhibit  also  sperm 

An  elegant  experiment  on  the  behaviour  of  cytochrome  in  sperma- 
tozoa was  performed  by  Rothschild  (1948a,  d)  who  demonstrated 
that  the  oxygen  uptake  of  sea-urchin  sperm  is  inhibited  by  carbon 
monoxide  and  that  the  inhibition  can  be  completely  reversed  by 
white  light  but  not  by  light  of  the  548  m^^  wavelength;  the  non- 
reversal  at  that  particular  wavelength  being  due  to  lack  of  absorp- 
tion by  cytochrome  oxidase  in  this  region  of  the  spectrum.  By 
interposing  between  the  source  of  light  and  the  microscope  a  colour 
filter  transmitting  light  of  the  548  m/n  wavelength,  Rothschild  was 
able  to  observe  spermatozoa  microscopically,  in  the  presence  of 

92  The  Biochemistry  of  Semen 

carbon  monoxide,  as  if  they  were  in  the  dark.  Under  these  condi- 
tions, he  found  that  carbon  monoxide  depressed  the  respiration 
without  a  corresponding  decrease  in  sperm  motiUty.  A  similar  con- 
clusion that  respiration  can  be  dissociated  from  motility  was  reached 
by  Robbie  (1948)  from  his  study  of  the  effect  of  cyanide  on  the 
spermatozoa  of  the  sand-dollar  {Echinarachnius  par  ma). 

The  cytochrome  system  of  sea-urchin  spermatozoa  includes  cyto- 
chrome e  and  in  this  respect,  it  differs  from  mammalian  sperm 
(Keilin  and  Hartree,  1949).  Starfish  {Asterias  forbesii)  spermatozoa 
on  the  other  hand,  exhibit  a  spectroscopic  pattern  of  cytochrome 
very  similar  to  sea-urchins  (Borei  and  Metz,  1951).  There  is  also 
some  evidence  that  cytochrome  occurs  in  plant  sperm.  This  follows 
from  the  observation  by  Rothschild  (1951c)  that  the  movements  of 
bracken  spermatozoids  {Pteridium  aquilinum  (L.)  Kuhn)  are  photo- 
reversibly  inhibited  by  carbon  monoxide. 

The  information  gained  by  Zittle  and  Zitin  (1942a)  from  experi- 
ments on  the  oxidation  of  /7-phenylenediamine  by  spermatozoa 
disintegrated  by  sonic  treatment,  coupled  with  earlier  observations 
on  the  indophenol  colour  reaction,  indicated  that  the  cytochrome 
system  is  located  in  the  cytoplasm  of  the  middle-piece  and  tail, 
rather  than  in  the  sperm-head.  Our  own  spectroscopic  studies  led  us 
to  the  same  conclusion;  the  examination  of  the  disintegrated  middle- 
pieces  and  tails  revealed  the  presence  of  all  three  cytochromes  with 
cytochrome  a  predominating;  these  'homogenates'  oxidized  rapidly 
both  /7-phenylenediamine  and  succinic  acid,  and  the  rate  of  oxygen 
consumption  could  be  substantially  increased  by  the  addition  of 
cytochrome  c. 


It  did  not  escape  Miescher's  notice  that  salmon  spermatozoa  differ 
from  other  cells  by  their  restricted  ability  to  decompose  hydrogen  per- 
oxide. Some  slight  catalase  activity  has  been  reported  in  mammaUan 
semen  (Shergin,  1940)  but  it  is  questionable  whether  this  was  due 
to  the  spermatozoa  themselves  or  to  some  accidental  contamination 
of  semen  with  blood,  pus  or  bacteria.  The  deficiency  of  catalase  in 
normal  and  cleanly  collected  bull  semen  is  in  fact,  so  typical  that 
Blom  and  Christensen  (1944,  1947)  base  on  it  a  method  for  rating 
the  'hygienic  quality'  of  bull  semen;  the  test  is  carried  out  in  Denmark 

Protein  Constituents  of  Spermatozoa  93 

in  special  'catalase  tubes'  in  whichi  hydrogen  peroxide  is  added  to 
semen  and  the  volume  of  evolved  oxygen  recorded.  In  ram  sperma- 
tozoa, even  after  mechanical  disintegration,  we  were  able  to  detect 
only  a  very  weak  catalase  activity:  an  extract  from  0-2  g.  sperm  (wet 
weight)  required  20  min.  at  18°  to  decompose  a  quantity  of  hydrogen 
peroxide  which  would  have  been  decomposed  in  2  min.  by  0001  ml. 
blood.  Sea-urchin  semen  on  the  other  hand,  contains  much  more 
catalase  (Evans,  1947;  Rothschild,  1948c,  1950c;  Barron,  Gasvoda 
and  Flood,  1949;  Rybak  and  Gustafson,  1952). 

The  lack  of  catalase  in  mammalian  semen  explains  the  harmful 
effects  of  hydrogen  peroxide  and  pure  oxygen  on  spermatozoa  (see 
p.  58).  It  is  also  of  considerable  physiological  interest  for  another 
reason,  inasmuch  as  the  spermatozoa  themselves  produce  hydrogen 
peroxide  in  vitro  during  the  oxidation  of  certain  amino-acids  (see 
p.  117). 

Hyaliironidase  and  other  'lytic'  agents 

The  term  'hyaluronidase'  in  its  widest  sense,  designates  the  muco- 
lytic enzyme,  or  rather  a  group  of  enzymes,  which  bring  about  the 
depolymerization  and  hydrolysis  of  hyaluronic  acid.  The  muco- 
polysaccharide called  hyaluronic  acid  is  a  polymer  of  the  disac- 
charide  hyalobiuronic  acid  which  consists  of  A^-acetylglucosamine 
and  D-glucuronic  acid;  its  enzymic  degradation,  that  is  depoly- 
merization and  hydrolysis,  is  believed  by  Meyer  and  his  school 
(1937,  1952)  to  be  due  to  the  opening  of  the  A^-acetylglucosaminidic 
bonds.  Thus  it  should  be  possible  to  assess  the  activity  of  hyalu- 
ronidase by  the  determination  of  the  reducing  groups  liberated  by 
the  enzymic  process.  In  actual  practice,  however,  this  is  only  possible 
with  the  use  of  purified  hyaluronidase  since  crude  enzyme  prepara- 
tions often  liberate  additional  reducing  groups  through  the  forma- 
tion of  free  glucuronic  acid  and  A^-acetylglucosamine  by  i^-glu- 
curonidase  and  /S-glucosaminidase,  respectively.  Apart  from  the 
'reductimetric'  method,  however,  there  are  several  other  ways  in 
which  the  activity  of  hyaluronidase  can  be  measured;  among  those 
in  use  is  the  'mucin  clot  prevention  (m.c.p.)  test'  in  which  the  preci- 
pitation by  acetic  acid  of  the  clot-like  protein-hyaluronic  acid 
complex  is  prevented  by  the  enzyme;  the  'turbidimetric'  method  is 
based  on  the  observation  that  purified  hyaluronate  at  pH  4-2,  gives 

94  The  Biochemistry  of  Semen 

a  fairly  stable  colloidal  suspension  with  dilute  serum,  whereas 
depolymerized  hyaluronate  under  identical  conditions  remains  clear; 
the  'viscosity  reduction  (v.r.)'  method  measures  the  decline  in  vis- 
cosity caused  by  depolymerization;  in  the  so-called  'Spinnbarkeit'- 
method  the  stringiness  of  hyaluronic  acid  is  assessed  by  means  of 
a  special  device,  before  and  after  enzymic  treatment.  Each  of  the 
above  methods,  however,  is  open  to  criticism  and  limited  in  its 
scope  (cf.  Lundquist,  1949a;  Swyer  and  Emmens,  1947;  Meyer  and 
Rapport,  1952). 

The  mammalian  testis  and  sperm  are  the  richest  animal  sources 
of  hyaluronidase.  The  existence  in  testes  and  spermatozoa  of  a 
'spreading'  or  'diffusing'  factor  which,  when  injected  intradermally, 
increases  the  permeability  of  the  skin  to  fluids,  was  established  by 
Hoff'man  and  Duran-Reynals  (1931)  and  McClean  (1930,  1931);  but 
Chain  and  Duthie  (1939,  1940)  deserve  the  credit  for  being  the  first 
to  show  that  purified  preparations  of  the  testicular  spreading  factor 
possess  strong  hyaluronidase  activity.  Their  finding  was  soon  con- 
firmed by  other  workers  who  made  several  attempts  to  purify  the 
enzyme  (Hahn,  1943;  Freeman,  Anderson,  Webster  and  Dorfman, 
1950;  Tint  and  Bogash,  1950).  The  best  preparations  of  bovine 
testicular  hyaluronidase  so  far  available  are  over  ten  thousand  times 
more  active  than  the  testicular  tissue  itself,  but  as  yet,  even  the  most 
highly  purified  enzyme  does  not  appear  to  be  a  homogeneous  protein. 

Hyaluronidase  originates  in  the  seminiferous  epithelium  of  the 
mature  testis,  and  in  semen  it  is  associated  with  the  spermatozoa  and 
not  with  the  seminal  plasma  (Werthessen,  Berman,  Greenburg  and 
Gargill,  1945;  Joel  and  Eichenberger,  1945;  Kurzrok,  Leonard  and 
Conrad,  1946;  Swyer,  1947^;  Jacquet,  Plessis  and  Cassou,  1951). 
The  content  of  hyaluronidase  per  sperm  cell  is  highest  in  rabbit  and 
bull;  there  is  less  of  it  in  human  and  boar  sperm,  very  little  in  dog, 
and  practically  none  in  birds  and  reptiles. 

Although  it  is  actually  a  part  of  the  sperm  cell,  hyaluronidase  is 
nevertheless  so  readily  released  by  spermatozoa  into  the  surround- 
ing medium  that  it  must  be  assumed  to  be  located  somewhere  very 
close  to  the  cell  surface,  possibly  on  the  sperm-head  (Hechter  and 
Hadidian,  1947;  Johnston  and  Mixner,  1947;  Perlman,  Leonard 
and  Kurzrok,  1948).  A  few  hours'  freezing  of  an  aqueous  sperm 
suspension  at  -10^,  or  24  hours'  standing  at  0°,  has  been  found  by 

Protein  Constituents  of  Spermatozoa  95 

Swyer  (1947«,  b)  to  be  quite  sufficient  for  hyaluronidase  to  pass 
completely  into  solution.  In  an  isotonic  medium  spermatozoa  also 
tend  to  liberate  hyaluronidase  but  when  the  concentration  of  the 
enzyme  outside  the  cell  reaches  a  certain  level  it  seems  to  prevent 
further  leakage.  Indeed,  under  certain  experimental  conditions, 
spermatozoa  depleted  of  hyaluronidase  have  been  found  to  be 
capable  of  reabsorbing  the  enzyme  from  a  hyaluronidase-rich 
medium  (Emmens  and  Swyer,  1948;  Swyer,  1951). 

The  physiological  role  of  sperm  hyaluronidase  is  far  from  clear 
at  present.  It  may  be  related  in  some  as  yet  unknown  manner, 
to  the  spsrmiogenic  function  of  the  testis,  but  there  is  also  soms 
indication  that  in  certain  mammals  at  any  rate,  hyaluronidase  acts 
by  facilitating  the  contact  between  the  male  and  female  gametes 
through  a  direct  liquefying  action  on  the  viscous  gel  which  cements 
the  follicle  cells  around  freshly  ovulated  eggs. 

Much  thought  has  been  devoted  in  the  past  to  the  problem  of  the 
participation  of  'lytic'  agents  in  the  process  of  ovum  fertilization, 
and  to  what  at  one  time  used  to  be  called  the  'ovulase'  activity  of 
spermatozoa.  However,  the  early  investigators  of  this  problem  were 
in  the  main  concerned  with  lower  animals.  In  many  molluscs, 
fishes  and  amphibia,  the  unfertilized  egg  is  normally  surrounded  by 
a  viscous  'jelly  coat'  and  a  membrane,  which  the  spermatozoon 
must  penetrate  before  fertiUzation  can  be  effected.  To  explain  this 
process,  several  investigators  postulated  the  presence  of  lytic  agents 
in  the  spermatozoa,  capable  of  mediating  the  fusion  of  the  gametes, 
but  there  has  been  little  evidence  that  these  agents  are  in  fact  enzymic 
in  nature,  until  Tyler's  (1939,  1942)  discovery  of  the  'egg-membrane 
lysin',  a  heat-labile  protein-enzyme  which  he  extracted  from  the 
sperm  of  two  molluscs,  the  key-hole  limpet  Megathura  cremilata 
and  the  abalone  Haliotis  cracherodii.  With  sperm  extracts  of  these 
species,  the  disappearance  of  the  egg  membrane  could  be  demon- 
strated within  about  3  min.,  if  the  gelatinous  coat  of  the  egg 
was  present,  and  in  less  than  30  sec,  if  the  coat  has  been  first 

The  occurrence  of  similar  lytic  enzymes  in  the  sperm  of  other 
lower  animals  is  still  under  discussion  (cf.  Tyler,  1948;  Berg,  1950; 
Runnstrom,  1951).  Several  lytic  agents  have  been  described  in  sea- 
urchin  spermatozoa.  One  of  them  is  the  previously  mentioned 

96  The  Biochemistry  of  Semen 

androgamone  III  ('egg-surface  liquefying  agent',  'sperm  lysin')  dis- 
covered by  Runnstrom  and  his  co-workers  and  shown  to  be  a  heat- 
stable  alcohol-soluble  substance,  probably  a  fatty  acid  (p.  71).  The 
other  is  the  protein-like  jelly-coat  'dissolving'  or  'precipitating' 
factor,  identical  with  Hartmann's  androgamone  II;  the  disappearance 
of  the  egg-jelly  under  the  influence  of  this  protein-factor  was  origin- 
ally described  by  Hartmann  and  his  colleagues  in  Arbacia  pustulosa, 
but  a  similar  phenomenon  was  later  observed  in  other  sea-urchin 
species  as  well  (Tyler  and  O'Melveny,  1941;  Monroy  and  Ruff'o, 
1947;  Kraus,  1950;  Vasseur,  1951;  Monroy  and  Tosi,  1952).  The 
suggestion  has  been  put  forward  that  the  agent  which  helps  the  sea- 
urchin  sperm  to  penetrate  the  jelly-coat,  is  a  mucopolysaccharase 
similar  even  though  not  identical,  with  hyaluronidase.  This  hypo- 
thesis, however,  is  in  want  of  experimental  support.  It  is  also  difficult 
as  yet,  to  assign  any  definite  role  in  fertilization  to  the  proteolytic 
gelatm-liquefying  enzyme  which  Lundblad  (1950)  extracted  from 
the  sperm  of  Arbacia  lixula  and  Paracentrotus  lividus. 

In  mammals,  the  existence  of  an  enzymic  'cumulus-dispersing 
factor'  was  first  brought  to  light  by  Yamane  (1935),  Pincus  (1935) 
and  Pincus  and  Enzmann  (1936)  who  showed  that  both  sperm 
suspensions  and  extracts  from  rabbit  spermatozoa,  brought  in 
contact  with  unfertilized  rabbit  ova,  can  disperse  within  a  short 
time  the  follicle  cells  of  the  cumulus  oophorus.  In  1942,  McClean 
and  Rowlands  discovered  that  hyaluronidase  which  they  obtained 
not  only  from  testes  or  spermatozoa,  but  also  from  snake  venom 
and  bacteria,  can  act  as  a  cumulus-dispersing  factor  by  liquefying 
the  viscous  gel  which  cements  the  follicle  cells  around  freshly  ovu- 
lated rat  ova.  Similar  results  on  the  mouse  were  reported  by  Fekete 
and  Duran-Reynals  (1943)  who  also  noted  that  the  gel  of  the 
cumulus  responds  to  metachromatic  staining  with  toluidine  blue 
like  hyaluronic  acid. 

It  remains  one  of  the  unsolved  mysteries  in  the  phenomenon  of 
fertilization  that  although  the  actual  fertilization  consists  ultimately 
of  the  fusion  of  a  single  spermatozoon  with  the  ovum,  this  can  take 
place  apparently  only  after  a  multitude  of  spermatozoa  have  reached 
the  site  of  fertilization.  Moreover,  the  denudation  of  the  ovum  from 
follicular  cells  has  also  been  claimed  to  require  the  presence  of 
numerous  spermatozoa.   According  to   McClean  and  Rowlands 

Protein  Constituents  of  Spermatozoa  97 

(1942)  they  are  needed  to  create  and  keep  up  a  sufficiently  high  con- 
centration of  hyaluronidase  to  permit  the  denudation  of  the  egg. 
This  hypothesis  was  put  to  the  test  by  Rowlands  (1944)  who  found 
that  it  is  possible  to  increase  the  fertilizing  capacity  of  a  subnormal 
number  of  rabbit  spermatozoa  by  the  addition  of  hyaluronidase  in 
the  form  of  extracts  from  whole  dilute  rabbit  semen.  Similar  results 
were  reported  by  Leonard  and  Kurzrok  (1945,  1946). 

In  view  of  all  this,  little  wonder  that  many  investigators  became 
attracted  by  the  possibility  of  the  therapeutic  application  of  hyalu- 
ronidase in  infertility.  In  fact,  several  enthusiastic  reports  appeared 
of  success  in  human  infertility  of  oligospermic  origin,  achieved  by 
the  addition  of  bovine  testicular  hyaluronidase  to  human  semen. 
However,  subsequent  investigations  failed  to  bear  out  the  claim 
that  hyaluronidase  can  enhance  the  fertilizing  capacity  of  sperma- 
tozoa. According  to  Chang  (1947Z),  1949)  the  earlier  positive  results 
obtained  with  extracts  from  whole  semen  should  be  attributed  not 
to  hyaluronidase  but  to  the  effect  of  the  seminal  plasma  as  such. 
Similarly,  the  concept  that  a  high  sperm  concentration  in  the  vicinity 
of  the  egg  is  needed  to  denude  the  ovum  from  its  cumulus,  has  been 
questioned  by  Leonard,  Perlman  and  Kurzrok  (1947),  Austin  (1948), 
and  Austin  and  Smiles  (1948)  who  demonstrated  clearly  that  sperma- 
tozoa can  in  fact  penetrate  rat  ova  which  are  still  enclosed  in  the 
cumulus.  Presumably,  the  hyaluronidase  charge  carried  by  the  sper- 
matozoa makes  it  possible  for  the  individual  sperm  to  'burrow'  its 
way  through  the  viscous  gel  which  cements  the  follicular  cells. 
This,  however,  need  not  necessarily  involve  the  dispersion  of  the 
cumulus,  which  process  is  probably  aided  by  the  mechanical  action 
of  the  ciUa  or  by  some  other  tubal  factor. 

Another  development  in  the  field  of  sperm  hyaluronidase  per- 
taining to  the  role  of  this  enzyme  in  fertilization,  has  been  the 
attempt  to  use  certain  inhibitors  of  hyaluronidase  as  systemic  con- 
traceptives. Among  the  inhibitors  of  hyaluronidase,  presumably 
competitive  in  nature,  are  several  derivatives  of  hyaluronic  acid 
obtained  by  acetylation  or  nitration,  also  heparin,  and  a  number 
of  other  anticoagulants  and  mucopolysaccharides,  including  a  sub- 
stance present  in  blood  serum  (Ferraro,  Costa  and  Pelegrini,  1948; 
Hadidian  and  Pirie,  1948;  Pincus,  Pirie  and  Chang,  1948;  Meyer 
and  Rapport,  1952).  Another  two  groups  of  inhibitors,  some  of 

98  The  Biochemistry  of  Semen 

which  act  irreversibly,  consist  of  heavy  metals  and  quinones,  includ- 
ing certain  quinoid  compounds  derived  from  flavonoids  (Beiler  and 
Martin,  1947,  1948;  Rodney  et  ai,  1950).  Much  interest  was  aroused 
some  time  ago  by  a  report  that  phosphorylated  hesperidine,  a 
potent  in  vitro  inhibitor  of  hyaluronidase,  can  act  as  an  'anti- 
fertility  factor'  when  administered  to  mice  and  human  beings; 
however,  attempts  to  substantiate  this  claim  have,  so  far,  been  un- 
successful (Martin  and  Beiler,  1952;  Sieve,  1952;  Martin,  1953; 
Chang  and  Pincus,  1953;  Thompson,  Sturtevant  and  Bird,  1953). 

Sperm  nucleoproteins 

In  the  mature  sperm  cell,  the  sperm  nucleus  fills  the  head  almost 
completely,  the  surrounding  cytoplasm  being  very  scanty.  This 
nucleus  consists  of  closely  packed  chromatin  embedded  in  a  rela- 
tively small  amount  of  nuclear  sap.  The  first  to  investigate  the 
chemistry  of  the  sperm  nucleus  was  Miescher  (1878,  1897)  whose 
pioneer  studies  laid  a  foundation  for  the  modern  developments  in 
the  chemistry  and  physiology  of  the  cell  nucleus,  the  nucleoproteins 
and  the  nucleic  acids.  As  a  result  of  Miescher's  brilliant  researches, 
continued  and  extended  by  such  investigators  as  Kossel,  Schmiede- 
berg,  Burian,  Levene,  Steudel,  Lynch,  Hammarsten,  Rasmussen  and 
Linderstr0m-Lang,  and  many  others,  it  is  now  generally  recognized 
that  the  chief  component  of  sperm  chromatin,  one  which  confers 
upon  the  paternal  (haploid)  chromosomes  their  functions  as  trans- 
mitters of  inheritance,  is  by  its  chemical  nature,  a  deoxyribonucleo- 
protein^  and  consists  of  deoxyribonucleic  acid  conjugated  with  cer- 
tain basic  nuclear  proteins  such  as  protamines  and  histones. 

Miescher  used  for  his  work  chiefly  salmon  spermatozoa  which  he 
obtained  mostly  by  stripping  the  live  fish.  A  considerable  advantage 
of  this  method  is  that  the  material  thus  obtained  consists  entirely  of 
ripe  spermatozoa  and  is  therefore  of  uniform  composition.  This 
useful  material,  however,  is  not  always  procurable  and  is  sometimes 
replaced  by  whole  excised  fish  testes  which  are  less  suitable  as  they 
may  contain  some  immature  spermatozoa  even  during  the  breeding 
season.  Miescher's  routine  procedure  was  to  remove  first  the 
sperm-tails  by  plasmolysis  with  water  or  weak  acetic  acid  and  then 
to  treat  the  washed  suspensions  of  sperm-heads  with  ethanol  and 
ether,  to  remove  the  lipids.  In  order  to  separate  sperm  nucleic 

Protein  Constituents  of  Spermatozoa  99 

acid  from  the  nuclear  protein,  Miescher  treated  the  Hpid-free 
material  first  with  cold  mineral  acid  (e.g.  0-25-0-5°/o  hydrochloric 
acid)  to  remove  the  protein,  and  then  with  sodium  hydroxide  'until 
the  mixture  tasted  distinctly  caustic  to  the  tongue',  to  extract  nucleic 
acid  from  the  residue. 

The  deoxyribonucleoproteins  in  the  spermatozoa  of  a  great  many 
fishes,  including  salmon,  belong  to  the  group  of  micleoprotamines 
which  can  be  extracted  from  the  sperm  nuclei  with  salt  solutions.  A 
convenient  method  for  such  an  extraction  and  purification  has  been 
described  by  Pollister  and  Mirsky  (1946).  Spermatozoa  of  the  brown 
trout,  Salmo  fario,  were  'homogenized'  with  1m  solution  of  sodium 
chloride  in  a  Waring  mixer  and  the  very  viscous  extract  poured  into 
six  volumes  of  water;  this  caused  the  precipitation  of  the  nucleo- 
protamine  in  the  form  of  long  strands,  so  fibrous  that  they  could 
be  wound  around  a  glass  rod  and  transferred  in  this  way  to  another 
container.  The  fibrous  material  was  dissolved  in  M-NaCl  and  re- 
precipitated  with  water,  and  then  washed  successively  with  65% 
ethanol,  hot  95%  ethanol,  and  finally  with  ether;  at  this  stage  it 
contained  about  6%  phosphorus  and  18%  nitrogen,  and  consisted 
of  deoxyribonucleic  acid  and  protamine  in  a  6  :  4  ratio.  When  this 
material  was  dissolved  in  M-NaCl  and  dialysed  against  M-NaCl,  the 
protamine  slowly  passed  through  the  cellophane  membrane  leaving 
behind  the  solution  of  deoxyribonucleic  acid.  On  pouring  this  solu- 
tion into  five  volumes  of  ethanol,  a  fibrous  precipitate  of  the  nucleic 
acid  was  obtained,  which,  after  drying,  had  a  content  of  8-97% 
phosphorus  and  14-47%  nitrogen.  This  composition  approaches 
the  theoretical  value  for  the  sodium  salt  of  deoxyribonucleic  acid, 
9-28%  phosphorus  and  15-58%  nitrogen. 

The  removal  of  protamine  from  nucleoprotamine  solutions  in- 
duces no  significant  change  in  viscosity.  The  high  viscosity  of  nucleo- 
protamine solutions  is  due  entirely  to  the  highly  polymerized  nucleic 
acid.  Deoxyribonucleic  acid  prepared,  for  instance,  from  herring 
spermatozoa,  has  a  molecular  weight  of  800,000;  each  molecule  of 
it  requires  some  100  molecules  of  protamine  to  form  a  molecule 
of  nucleoprotamine.  According  to  some  calculations  by  Felix  (1952), 
a  single  fish  sperm  nucleus  contains  4-5x10^  nucleoprotamine 
molecules,  enough  to  provide  about  190,000  molecules  per  each 
chromosome,  or  several  thousand  per  each  gene. 

100  The  Biochemistry  of  Semen 

Unfortunately  however,  the  extraction  with  M-NaCl  is  not  a 
universal  means  for  the  separation  of  sperm  nucleoproteins.  In  the 
key-hole  limpet  or  freshwater  clam,  the  sperm  nucleoprotein 
resists  extraction  with  M-NaCl,  but  can  be  brought  into  solution 
with  a  2m  salt  solution,  whereas  no  nucleoprotein  can  be  extracted 
with  NaCl  of  either  concentration  from  the  sperm  of  man,  bull, 
boar  or  ram.  Moreover,  dialysis  against  M-NaCl  or  extraction  with 
dilute  mineral  acids  both  prove  inadequate  for  the  removal  of 
nuclear  proteins  from  mammalian  spermatozoa.  In  such  cases,  the 
separation  of  protein  can  be  brought  about  with  a  chloroform- 
octanol  mixture  (Sevag,  Lackman  and  Smolens,  1938),  but  before 
this  is  applied  it  is  necessary  to  separate  the  sperm  nucleus  from  the 
remainder  of  the  sperm  cell  by  ultrasonic  or  mechanical  treatment. 

Deoxyribonucleic  acid 

This  when  freed  from  nuclear  protein,  is  composed  of  mono- 
nucleotides, each  consisting  of  one  molecule  of  phosphoric  acid, 
one  molecule  of  the  sugar  D(-)2-deoxyribose,  and  one  molecule  of 
a  purine  or  pyrimidine  base:  adenine,  guanine,  cytosine  or  thymine. 
A  small  amount  of  yet  another  base,  5-methylcytosine  (Wyatt,  1950, 
1951),  has  been  found  so  far  in  the  sperm  deoxyribonucleic  acid  of 
man,  bull,  ram,  herring  and  sea-urchin  {Echinus  esculentus),  but 
probably  it  occurs  also  in  other  species. 

In  all  species,  deoxyribonucleic  acid  is  confined  entirely  to  the 
sperm  nucleus  as  can  be  demonstrated  by  various  staining  methods, 
and  particularly  by  the  'Feulgen  nucleal  reaction'.  This  reaction  was 
described  by  Feulgen  (1914,  1917)  at  first  as  a  colour  test  for  thymo- 
nucleic  acid,  but  later  it  was  adapted  for  the  staining  of  cell  nuclei 
(Feulgen  and  Rosenbeck,  1924). 

With  thymonucleic  acid  itself,  the  test  is  carried  out  best  on  a 
solution  of  sodium  nucleinate  (01  g./l  ml.)  prepared  in  a  boiling 
tube  on  the  water-bath.  The  solution  is  treated  with  1  ml.  2N-H2SO4, 
and  left  at  100°  for  3  minutes,  then  cooled  and  neutralized.  When  a 
drop  of  the  hydrolysate  is  mixed  with  a  few  ml.  of  Schiff^'s  fuchsin- 
sulphurous  acid  reagent  (a  0-5%  solution  of  fuchsin  decolorized 
with  SO2  and  the  excess  of  SO2  removed  by  suction),  an  intense 
purple  colour  develops.  The  chemistry  of  the  Feulgen  colour  re- 
action is  as  yet  only  partly  understood  but  is  believed  to  involve  the 

Protein  Constituents  of  Spermatozoa  101 

NHo  OH  NH2 

N  C  X  N  C  X  HN  CH 

I  II  CH  I  II  CH  I  II 

HC  C  /  H.,N— C  C  /  OC  CH 

^N^  ^NH  ^N^     ^NH  ^NH 

Adenine  Guanine  Cytosine 

(6-aminopurine)  (2-amino-6-hydroxy-  (2-hydroxy-6- 

purine)  aminopyrimidine) 

O  NH2 

II  I 

/^\  ^^\ 

HN  C— CH3  N  C— CH3 

I  II  I  II 


^NH  ^NH 

Thymine  5-Methyl-cytosine 

(5-methyl-2  :  6-dihydroxy- 

following  steps:  acid  hydrolysis  which  splits  off  purine  and  exposes 
deoxyribose;  transformation  of  deoxyribose  into  co-la  evuUnic  alde- 
hyde; lastly,  formation  of  a  purple-coloured  reaction  product  of 
this  labile  aldehyde  with  fuchsin-sulphurous  acid.  The  mechanism 
of  the  Feulgen  reaction  as  exhibited  by  the  cell  nuclei  in  histological 
preparations,  appears  to  be  even  more  involved  (Danielli,  1947; 
Davidson,  1950).  In  cellular  material,  however  carefully  carried  out, 
the  Feulgen  reaction  cannot  be  expected  to  yield  results  as  accurate 
and  specific  as  the  colour  test  with  pure  deoxyribonucleic  acid. 
Some  of  the  difficulties  inherent  in  the  application  of  the  reaction 
as  a  staining  method  for  sperm  nuclei,  have  been  pointed  out  by 
Feulgen  and  Rosenbeck  themselves,  who  noted  that  when  fresh 
smears  of  human  semen  were  treated  with  Schiff 's  fuchsin-sulphurous 
acid  reagent,  the  sperm-heads  stained  rather  weakly,  whereas 
the  middle-pieces  and  tails,  though  devoid  of  nuclear  material, 
stained  strongly.  This  observation  was  followed  by  a  demonstration 
that  the  fuchsin-staining  material  present  in  the  middle-piece  and 
tail  is  a  lipid,  'plasmal',  which  can  be  distinguished  from  the 

102  The  Biochemistry  of  Semen 

nucleoprotein  of  the  sperm-head  by  its  solubiUty  in  ethanol.  Similar 
observations  with  animal  sperm  were  later  reported  by  other  investi- 
gators. There  is  no  doubt,  however,  that  if  carried  out  properly  and 
under  conditions  which  eliminate  interference  from  other  fuchsin- 
staining  substances,  the  Feulgen  nucleal  reaction  can  be  made 
specific  for  the  sperm  nucleus.  Several  authors  have  stressed  the 
fact  that  the  base  and  the  posterior  region  of  the  sperm-head  stains 
particularly  strongly  (Marza,  1930;  Wislocki,  1950;  Friedlaender 
and  Fraser,  1952). 

Although  present  in  the  sperm  nuclei  of  all  species,  the  composi- 
tion of  deoxyribonucleic  acid  varies  somewhat  from  one  species  to 
another,  with  regard  to  the  proportion  of  the  various  purine  and 
pyrimidine  bases.  But  within  any  given  species,  all  body  cells,  includ- 
ing the  spermatozoa,  seem  to  yield  on  purification  the  same  nucleic 
acid,  that  is  one  with  the  same  molar  ratio  of  adenine,  guanine, 
cytosine  and  thymine.  In  the  species  so  far  examined,  the  ratio  of 
adenine  to  thymine,  of  guanine  to  cytosine,  and  of  total  purines  to 
total  pyrimidines,  was  shown  to  equal  unity. 

The  results  of  the  analysis  of  bases  in  deoxyribonucleic  acid  from 
ram  spermatozoa  are  shown  in  Plate  V.  The  spermatozoa  were  dis- 
integrated mechanically  and  suspensions  of  washed,  tail-free  sperm- 
heads  were  prepared  (Mann,  \95\b).  Nucleic  acid  was  separated 
from  the  nuclear  proteins  of  the  sperm-heads  by  the  chloroform- 
octanol  treatment,  hydrolysed  with  formic  acid,  and  the  liberated 
bases  separated  on  paper  chromatograms  by  Wyatt's  method,  using 
as  solvent  an  aqueous  solution  containing  65%  /^(^propanol  and 
2N-HC1.  The  bases  set  free  by  acid  hydrolysis  and  determined 
spectrophotometrically  according  to  Markham  and  Smith  (1949) 
were:  guanine,  adenine,  cytosine  and  thymine  in  a  molar  ratio  of 
0-91  :  M3  : 0-86  :  110.  In  addition,  there  was  a  small  amount  of 
methylcytosine  which  in  Plate  V  is  only  just  visible  as  a  faint  ultra- 
violet-absorbing band  below  cytosine;  the  molar  ratio  of  cytosine 
to  methylcytosine  was  1  :  005. 

Deoxyribonucleic  acid  (Na-salt)  of  human  sperm  was  studied  by 
Chargaff  and  his  colleagues  (Chargaff,  Zamenhof  and  Green,  1950; 
ChargafF,  1951a,  b)\  it  contains  16%  nitrogen  and  8-9%  phosphorus 
(Zamenhof,  Shettles  and  Chargaff,  1950);  the  specific  viscosity  of  a 
0-135%  solution  in  water  equals  7-0,  and  the  sedimentation  constant 


SPERM    HEADS   (RAM)   :     DRNA 


Guanine        0.9I 

Adenine        |.|3 

Cytosjne      0.86 


Thymine        t.lO 


Contact  print,  taken  with  ultraviolet  light,  of  a  paper  chromatogram 
from  the  acid  hydrolysate  of  ram  sperm-heads.  The  figures  indicate 
the  molar  ratios. 

Protein  Constituents  of  Spermatozoa  1 03 

(Soo)  for  a  0-22%  solution  in  0-2M-NaCl  is  5-7,  a  value  which  re- 
sembles closely  that  given  by  an  undegraded  specimen  of  calf 
thymus  nucleic  acid.  According  to  Elmes,  Smith  and  White  (1952), 
the  molar  proportions  of  the  purine  and  pyrmidine  bases  in  deoxy- 
ribonucleic acid  from  human  sperm  and  tissues  are:  guanine  0-92 
(standard  error  of  observations  0-036),  adenine  1-23  (0-068),  cyto- 
sine  0-84  (0077),  thymine  1-01  (0-09)  and  5-methylcytosine  0-03. 

Table  14.  Salmon  sperm  deoxyribonucleic  acid  (Chargaff  et  al,  1951) 

(Molar  ratios  between  the  bases.) 

Purines  to  pyrimidines  1-02 

Adenine  to  thymine  1-02 

Guanine  to  cytosine  1-02 

Adenine  to  guanine  1-43 

Thymine  to  cytosine  1  -43 

The  analysis  of  the  nucleic  acid  obtained  from  the  sperm  nucleo- 
protamine  of  salmon,  Salmo  salar  (Chargaff,  Lipschitz,  Green  and 
Hodes,  1951)  gives  a  good  illustration  of  the  regularity  in  the  com- 
position of  deoxyribonucleic  acid.  The  nucleoprotamine  was  pre- 
pared by  extraction  with  a  salt  solution,  the  nucleic  acid  set  free  by 
chloroform-octanol  treatment,  and  after  some  further  purification, 
recovered  as  the  sodium  salt.  Two  specimens  were  isolated  in  this 
way,  containing  14-3%  nitrogen  and  8-9%  phosphorus  and  14-8% 
nitrogen  and  8-9%  phosphorus,  respectively.  The  ultraviolet  absorp- 
tion spectrum  measured  in  M-phosphate  buffer  pH  7-1,  exhibited 
a  maximum  at  260  mi-i.  The  specific  viscosity  in  distilled  water  at 
30-3°,  was  found  to  be  29-6  for  a  0-22%  solution,  and  5-6  and  20 
for  0-11  and  0-055%  solutions,  respectively.  The  bases  were  set 
free  by  acid  hydrolysis  and  analysed  chromatographically  and 
spectroscopically.  The  ratios  of  the  purine  to  the  pyrimidine  bases, 
of  adenine  to  thymine,  and  of  guanine  to  cytosine  equalled  unity; 
adenine  and  thymine  exceeded  guanine  and  cytosine  by  about  40% 
(Table  14). 

Not  only  the  composition  but  also  the  content  of  deoxyribonucleic 
acid  in  the  cell  nucleus  appears  to  be  fairly  constant  and  characteris- 
tic for  each  animal  species.  The  haploid  nucleus  of  the  sperm  cell, 
however,  differs  from  the  diploid  nuclei  of  the  somatic  cells  in  that 
it  contains  a  reduced  amount  of  chromatin  and  consequently  it  is 


The  Biochemistry  of  Semen 

assumed  to  possess  only  one-half  of  the  amount  of  deoxyribonucleic 
acid  present  in  the  somatic  nuclei  of  a  given  species  (Boivin,  Vendrely 
and  Vendrely,  1948;  Vendrely  and  Vendrely,  1948,  1949,  1952; 
Mirsky  and  Ris,  1949,  1951;  a  review  by  Colette  Vendrely,  1952). 
To  illustrate  this  point,  Table  1 5  gives  data  on  the  content  of  deoxy- 
ribonucleic acid  in  the  sperm  (haploid)  nuclei  and  in  the  somatic 
(diploid  or  polyploid)  cell  nuclei  of  various  species.  The  values 
range  from  0- 1 1  x  10~^  mg.  for  the  diploid  nucleus  of  a  sponge  cell  to 
168x10"^  for  a  diploid  cell  nucleus  of  Amphiuma,  a  urodele;  the 
nucleic  acid  in  the  spermatozoa  of  the  carp,  trout,  pike,  tench,  toad, 
cock  and  bull,  is  seen  to  be  approximately  one-half  the  content  of 
the  somatic  cell  nuclei.  A  high  proportion  of  the  data  listed  in 
Table  15,  were  obtained  by  means  of  the  analytical  procedures 

Table  15.  Deoxyribonucleic  acid  content  of  single  nuclei  in 
somatic  cells  and  spermatozoa 

(Contents  expressed  in  mg.  x  10~^  deoxyribonucleic  acid  per  cell  nucleus; 
figures  in  brackets  refer  to  authors.) 


Type  of  cell    mg. 

X 10- Vnucleus 


Orange  sponge,  Dysidea  crawshagi 




Jelly  fish,  Cassiopeia 


0-33  (3) 


Sea-urchin,  Arbacia 




Sea-cucumbsr,  Stichopus  diabole 


0-67  (5) 
0-70  (5) 
0-98  (3) 
0-90  (3) 
0-90  (3) 


Limpet,  Fisswella  barbadensis 
Snail,  Tec  tar  ins  muricatus 


0-50  (3) 
0-67  (3) 


Cliff  crab,  Plagiisia  depressa 
Goose  barnacle 




Sturgeon,  Acipenser  stiirio 
Carp,  Cyprinus  carpio 


3-2    (3) 
3-2    (6) 
1-6    (8) 

Protein  Constituents  of  Spermatozoa 


Trout,  Salmo  ir ideas  Gibb. 

Salmo  fario 
Pike,  Esox  lucius 
Tench,  Tinea  tinea 






Green  turtle 


Domestic  fowl 







(1)  Davidson  et  al.  (1950);  (2)  Mirsky  and  Ris  (1949);  (3)  Mirsky  and 
Ris  (1951);  (4)  Vendrely  and  Vendrely  (1948);  (5)  Vendrely  and  Vendrely 
(1949);  (6)  Vendrely  and  Vendrely  (1952);  (7)  Vendrely  (1952);  (8)  Vendrely 
and  Vendrely  (1953). 


4-9    (6) 


2-45  (6) 


5-79  (2) 


2-67  (2) 




0-85  (6) 




0-85  (6) 


168  (3) 


15  (3) 


7-33  (2) 


3-70  (2) 


4-92  (3) 


2-34  (2) 


f2-56  (1) 
12-39  (2) 


2-54  (1) 


2-20  (1) 


2-45  (1) 

Cock  sperm 



2-3    (7) 


2-1    (7) 

r6-4   (4) 


<^6-2    (2) 

l8-4    (3) 


5-9    (4) 


6-9    (4) 


6-4    (4) 

Bull  sperm 

r3-3  (4) 
\2-82  (2) 


5-0    (7) 


5-2    (7) 


r6-l    (7) 

15-4    (7) 


50    (7) 


5-3    (7) 


5-9    (7) 

106  The  Biochemistry  of  Semen 

developed  by  Schmidt  and  Thannhauser  (1945)  and  Schneider 
(1945,  1946)  which  are  based  largely  on  determinations  of  phos- 
phorus and  involve  the  removal  of  (i)  the  'acid-soluble  phosphorus 
compounds'  (extraction  with  cold  trichloroacetic  acid),  and  (ii)  the 
phospholipids  (extraction  with  ethanol  and  ether),  prior  to  the 
analysis  of  nucleic  acid  (see  also  Table  16). 

The  basic  nuclear  proteins,  protamines  and  histones 

The  proteins  conjugated  with  deoxyribonucleic  acid  are  of  the 
basic  type  and  have  been  shown  to  be  either  protamines  or  histones 
in  most  instances  so  far  examined.  Protamines  have  been  isolated 
from  fish  spermatozoa  of  several  species.  Of  the  various  protamines, 
the  best  known  are  salmine  from  salmon  and  trout  sperm,  and 
clupeine  from  herring  sperm.  Much  less  is  known  about  the  other 
protamines,  such  as  scombrine  (mackerel),  cyclopterine  (lump- 
sucker),  esocine  (pike),  thynnine  (tunny  fish),  percine  (perch), 
cyprinine  (carp)  and  st urine  (sturgeon). 

Judging  from  the  molecular  weight  and  amino  acid  composition, 
salmine,  clupeine  and  scombrine  have  a  relatively  simple  structure, 
there  being  approximately  two  molecules  of  arginine  to  one  molecule 
of  monoamino  acid.  Thus,  for  example,  the  analysis  of  salmine 
sulphate  prepared  from  the  spermatozoa  of  the  Spring  or  Chum 
salmon  (Tristram,  1947,  1949)  suggests  a  molecular  weight  of  about 
8000,  with  a  total  of  58  amino  acid  residues:  40  arginine,  8  isoleu- 
cine,  2  valine,  4  proline,  3  glycine,  1  alanine  and  7  serine;  this  sal- 
mine sulphate  contains  19-85%  sulphuric  acid,  i.e.  40  equivalents 
per  molecule,  sufficient  to  combine  with  all  arginine  residues. 
Another  salmine  sulphate,  one  prepared  from  the  sperm  of  the 
Columbia  River  salmon  (Block  and  Boiling,  1945)  is  said  to  con- 
tain 67  amino  acid  residues:  47  arginine,  1  isoleucine,  3  valine, 
6  proline,  4  alanine  and  6  serine  (see  also:  Corfield  and  Robson, 

The  results  of  formol  titration  indicate  that  salmine  contains  one 
free  amino  or  imino  group  per  molecule  of  8000,  and  the  end  group 
assay  carried  out  by  means  of  the  dinitrofluorobenzene  method 
(Sanger,  1952)  suggests  that  the  N-terminal  position  is  occupied  by 
the  imino  group  of  proline.  In  clupeine,  the  A^-terminal  position  is 
also  taken  up  by  proline,  whereas  at  the  other  end  of  the  amino  acid 

Table  16.  Distribution  of  phosphorus  compounds  in  ram  semen 

(Results  based  on  analysis  of  16-8  ml.  ram  semen,  representing  14 
ejaculates  from  7  rams;  average  volume  of  single  ejaculate  1-2  ml.; 
3,050,000  spermatozoa/yal.  Semen  separated  by  centrifugation  for  20  min. 
at  10,000^,  into  sperm,  26%  v/v,  and  seminal  plasma,  74%  v/v.) 

mg.  P/100  ml.  semen 

In  whole  In  sperm        In  seminal 

semen  plasma 

Total  phosphorus  328-5  186-7  141-8 

Acid-soluble  phosphorus  (in  trichloro- 
acetic acid  extract) 

Pjnorg  (orthophosphate  determined  as 

MgNH4P04)  10-3  3-1  7-2 

Po  (phosphate  reacting  directly  with 
molybdate)  11-9  4-7  7-2 

P7  (phosphorus  which  appears  as  ortho- 
phosphate  after  7  min.  hydrolysis 
withN-HCl)  15-4  8-1  7-3 

P30  (phosphorus  which  appears  as 
orthophosphate  after  30  min.  hydro- 
lysis with  N-HCl)  15-9  8-6  7-3 

Ptot.  ac.  sol.  (total  acid-soluble  phosphate 

determined  after  incineration)  159-4  27-4  132-0 

Patp  (labile  phosphate  of  adeno- 
sine triphosphate  determined  in  the 
Ba-salt  by  the  method  of  Parnas 
and  Lutwak-Mann,  1935)  4-3  4-3  00 

Phexose  (phosphate  of  6-phosphohexose 
determined  in  the  supernatant  from 
Ba-ATP)  6-2  60  0-2 

Phospholipid  phosphorus  (extracted  with 
ethanol  and  ether  from  the  residue 
insoluble  in  trichloroacetic  acid)  30-8  27-9  2-9 

Deoxyribonucleic  acid-phosphorus  (ex- 
tracted with  KOH  from  the  residue 
left  after  removal  of  acid-soluble  P 
and  phospholipid;  precipitated  from 
the  KOH  extract  by  acid)  1110  1110*  00 

Residual  phosphorus  (left  after  removal 
of  acid-soluble  compounds,  phos- 
pholipids and  deoxyribonucleic  acid)       27-3  20-4  6-9 

*  Corresponding  to  3-2  x  10~^  mg.  deoxyribonucleic  acid/sperm  cell. 


108  The  Biochemistry  of  Semen 

chain,  the  C-terminal  position  is  filled  by  arginyl-arginine  (Dirr  and 
Felix,  1932).  Arginine-arginine  linkages  occur  also  with  great  fre- 
quency in  the  amino  acid  chain  itself  (Felix  and  Schuberth,  1942). 
The  following  peptides  have  been  identified  as  breakdown  products 
of  a  partial  hydrolysis  of  clupeine:  arginyl-arginine,  triarginyl- 
arginine,  alanyl-arginyl-arginine,  seryl-arginyl-arginine,  and  alanyl- 
alanine;  on  the  basis  of  the  available  evidence,  Felix,  Fischer  and 
Krekels  (1952)  suggested  the  following  sequence  of  proline  (Prol.), 
arginine  (Arg.)  and  monoamino  acids  (M.)  in  clupeine, 

Prol.  (Arg.  Arg.  Arg.  Arg.  M.  M.)^  Arg.  Arg. 

Salmine  and  clupeine,  irrespective  of  their  origin,  both  contain 
arginine  as  the  sole  basic  amino  acid;  in  the  corresponding  nucleo- 
protamines  the  ratio  between  the  arginine  residues  of  the  protamines 
and  the  phosphoric  acid  equivalents  of  nucleic  acid  is  not  far  from 
unity,  usually  about  0-95  (Felix,  1951).  In  other  protamines,  on  the 
other  hand,  e.g.  percine  and  sturine,  a  certain  proportion  of  the 
basic  units  is  present  in  the  form  of  histidine  and  lysine. 

On  close  inspection  of  analytical  data  relating  to  the  various  pro- 
tamines, there  stands  out  a  considerable  degree  of  variability  in  the 
amino  acid  composition,  even  in  closely  related  species.  The  situa- 
tion is  even  more  complicated  in  sperm  nucleoproteins  which  con- 
tain histones  instead  of  protamines.  The  histones  such  as  occur  for 
instance,  in  the  sperm  nucleoproteins  of  sea-urchins  {arbacine  of 
Mathews,  1897)  and  cod-fishes  (gadushistone  and  lotahistone)  are 
characterized  by  a  wider  range  of  amino  acids,  including  tyrosine. 
It  is  not  improbable  that  some  of  the  observed  variations  in  the 
amino  acid  composition  of  protamines  and  histones,  represent  dis- 
tinct species  characteristics  analogous  to  those  which  are  encoun- 
tered in  other  proteins,  e.g.  in  the  globins  of  various  haemoglobins. 
On  the  other  hand,  however,  the  standards  for  the  assessment  of 
chemical  purity  of  nuclear  proteins  are  open  to  criticism,  and  it  is  un- 
certain whether  the  examined  protamines  and  histones  were  always 
really  pure.  Quite  likely  a  sperm  nucleus  may  contain  in  some 
cases  more  than  one  basic  protein,  that  is,  a  main  protamine  or 
histone,  together  with  a  smaller  amount  of  a  'subsidiary'  product 
(Stedman  and  Stedman,  1951).  Furthermore,  there  is  also  the  pos- 
sibility that  some  of  the  reported  differences  in  the  composition  of 

Protein  Constituents  of  Spermatozoa  109 

nuclear  proteins  are  simply  due  to  the  use  of  material  containing 
variable  proportions  of  mature  and  immature  spermatozoa.  Sperma- 
tocytic  development  is  well  known  to  be  associated  with  characteris- 
tic changes  in  the  amino  acid  composition  of  the  nuclear  proteins. 
Immature  spermatozoa  of  salmon,  for  instance,  obtained  directly 
from  excised  testes,  contain  a  histone  instead  of  the  salmine.  Simi- 
larly, the  testes  of  the  mackerel  yield  on  extraction  with  dilute 
hydrochloric  acid  a  histone,  'scombron',  instead  of  the  protamine 
'scombrine'.  This  and  similar  observations,  prompted  Kossel  (1928) 
to  acclaim  the  histones  as  'intermediary  stages'  in  the  transformation 
of  complex  proteins  into  protamines. 

77?^  non-basic  nuclear  proteins;  karyogen  and  chromosomin 

In  addition  to  the  basic  proteins,  the  sperm  nucleus  always  con- 
tains some  non-basic  or  so-called  residual  proteins.  In  lipid-free 
preparations  from  salmon  sperm-heads,  Miescher  found  19-78% 
protamine  (extracted  with  0-25-0-5%  HCl),  2-94%  acid-soluble  pro- 
tein material  other  than  protamine,  60-50%  of  nucleic  acid  (ex- 
tracted with  NaOH)  and  16-78%  of  an  iron-containing  residue 
which  was  insoluble  either  in  acid  or  in  alkali  and  which  he  believed 
to  contain  'karyogen',  the  'inner-space  protein  substance'  {Innen- 
raumsubstanz),  of  the  sperm  nucleus. 

It  was  found  later  that  the  residual  or  non-basic  nuclear  proteins, 
unlike  the  protamines  and  histones,  contain  tryptophan  as  a 
characteristic  component.  Opinions  are  divided,  however,  on  the 
problem  of  the  actual  ratio  of  non-basic  to  basic  proteins  in  the 
sperm  nucleus.  According  to  the  Stedmans  (1943,  1947),  the  nucleo- 
protamine  present  in  the  sperm-heads  of  salmon  accounts  for  no 
more  than  70%  of  the  dry,  lipid-free  material,  whereas  the  remainder 
is  made  up  largely  of  a  non-basic  protein  'chromosomin',  which 
contains  tryptophan.*  On  the  other  hand,  PoUister  and  Mirsky 
(1946)  state  that  the  nucleoprotamine  present  in  trout  spermatozoa 

*  The  Stedmans'  chromosomin  must  not  be  confused  with  chromosin, 
a  name  given  by  Mirsky  and  Pollister  (1946)  to  a  complex  extracted  with 
M-NaCl  from  isolated  cell  nuclei  of  various  organs,  including  thymus, 
liver,  spleen,  pancreas,  brain,  frog  testes  and  bacteria.  This  complex 
is  composed  of  deoxyribonucleic  acid,  histone,  and  a  tryptophan-contain- 
ing  protein. 

1 10  The  Biochemistry  of  Semen 

accounts  for  as  much  as  91%  of  the  dry,  lipid-free  mass  of  the  head 
nuclei.  These  authors,  however,  also  find  in  the  nuclei  a  charac- 
teristic tryptophan-containing  residual  protein. 

Keratin-like  protein  of  the  sperm  membrane 

Within  the  category  of  'residual'  sperm  proteins  are  also  certain 
highly  insoluble  sulphur-rich  proteins,  obtained  from  mammalian 
spermatozoa  and  derived  probably  from  the  sperm  membrane.  The 
first  mention  of  a  'sulphur-rich  substance,  containing  more  than 
4%  S  in  the  heads  of  bull  spermatozoa',  was  made  in  1878  by 
Miescher,  who  did  not,  however,  investigate  its  origin  and  compo- 
sition. Green  (1940)  extracted  ram  spermatozoa  successively  with 
dilute  acid  and  alkali,  and  obtained  a  residue  containing  19-3% 
nitrogen  and  11-4%  cystine,  which  he  believed  to  represent  the 
sperm  membrane.  The  possibility  of  a  keratin-like  protein  present 
in  the  sperm  membrane  is  strengthened  by  the  observations  of  Zittle 
and  O'Dell  (1941a)  on  the  solubilizing  action  of  thioglycolic  acid 
and  trimethylbenzylammonium  hydroxide  on  bull  spermatozoa.  In 
boar  sperm,  the  portion  which  remains  undissolved  after  prolonged 
treatment  with  N-NaOH,  consists  of  'ghost'  sperm-heads  which 
resemble  in  shape  the  sperm  membranes  (Thomas  and  Mayer,  1949). 
A  remarkable  property  of  the  sperm  membrane  which  can  be  demon- 
strated microscopically,  is  the  extraordinary  elasticity  of  the  sperm- 
head  structure;  this  was  convincingly  demonstrated  in  the  experi- 
ments of  Moench  and  Holt  (1929-32),  who  were  able  to  hook  the 
head  of  a  human  spermatozoon  with  a  microsurgical  needle  and 
to  stretch  it  very  considerably. 


Protein  Constituents  and  Enzymes 
of  the  Seminal  Plasma 

Proteoses  and  free  amino  acids.  Fibrinolysin  and  fibrinogenase.  Pepsino- 
gen. Ammonia  formation.  Amino  acid  oxidase.  Seminal  phosphatases; 
*acid'  and  'alkaline'  phosphatase;  5-nucleotidase;  pyrophosphatase. 
Enzymic  hydrolysis  of  adenosine  triphosphate. 

Proteoses  and  free  amino  acids 

A  DISCUSSION  of  the  nature  of  extracellular  protein  constituents  of 
semen  demands  the  recognition  of  certain  circumstances  which  are 
peculiar  to  the  seminal  plasma.  An  important,  though  sadly  ne- 
glected fact  is  that  the  protein  content  of  the  seminal  plasma  does 
not  remain  constant  after  ejaculation  but  undergoes  rapid  changes 
of  enzymic  character  which  manifest  themselves  in  a  progressive 
decrease  in  the  concentration  of  non-dialysable  protein-nitrogen 
and  a  simultaneous  accumulation  of  non-protein  nitrogen,  free 
amino  acids  and,  at  a  late  stage,  of  free  ammonia.  Unless  this  is 
fully  taken  into  account,  results  of  protein  and  amino  acid  analysis 
in  semen  are  but  of  little  significance  and  yield  no  information  on  the 
initial  distribution  of  nitrogenous  compounds.  This  applies  especi- 
ally to  human  semen  as  was  convincingly  demonstrated  by  Lund- 
quist  (1949c,  1952),  and  also  by  Jacobsson  (1950,  Fig.  11). 

Even  in  freshly  collected  seminal  plasma  a  large  proportion  of 
total  nitrogen  is  found  partly  as  a  protein-like  material  which  passes 
readily  through  semi-permeable  membranes  but  is  not  heat-coagu- 
lable  and  not  precipitated  by  trichloroacetic  acid;  accordingly,  this 
has  been  classified  as  propeptone,  hemialbumose,  primary  proteose, 
and  secondary  proteose  (thioalbumose  and  synalbumose)  (Posner, 
1888,  1892;  Marshall,  1922;  Goldblatt,  1935^).  In  human  semmal 
plasma,  out  of  a  total  content  of  about  3-5  to  5-5  g.  protein-like 
material  per  100  ml.,  no  more  than  18%,  usually  much  less,  is 
coagulated   by   heat,   and  about   60%   passes  through  cellulose 



The  Biochemistry  of  Semen 

membranes  which  are  impermeable  to  blood  serum  proteins 
(Huggins,  Scott  and  Heinen,  1942).  The  electrophoretic  pattern  of 
the  non-dialysable  portion  from  five  different  specimens  of  human 
seminal  plasma  has  been  examined  by  Gray  and  Huggins  (1942)  who 
observed  four  distinct  components  which  corresponded  to  serum 
albumin  (17-7-22-7%),  a-globulin  (19-8-27-8%),  i5-globulin  (34-3- 
44-5%),  and  y-globulin  (11 -4-21  0%).  Ross,  Moore  and  Miller 





"E  200 



2    I50 






^,— 0NH2-N 



20        40         60 
Minutes  after  ejaculation 

Fig.  1 1 .  Increase  of  non-protein  nitrogen  and  amino-nitrogen  content  in 

human  semen  on  incubation  at  37°. 

(Jacobsson,  1950) 

(1942)  also  carried  out  an  electrophoretic  and  chemical  analysis 
of  human  seminal  plasma  in  which  they  distinguished  five  protein 
fractions:  albumin  (less  than  002%);  'nucleoprotein'  (less  than 
004%);  proteose  ('Pi'),  which  was  not  heat-coagulable  and  passed 
through  a  membrane  of  25  A  pore  diameter;  two  water-insoluble 
proteins  ('Pg'  and  'P3')  and  a  mucoprotein  ('P4')  which  contained 
9-3%  N,  10-8%  hexosamine,  and  gave  on  hydrolysis  with  n-HCI 
reducing  substances  (26-8%)  but  no  uronic  acid.  The  strongly 
positive  periodic  acid-Schifif  reaction  which  is  very  characteristic 

Protein  Constituents  and  Enzymes  of  Seminal  Plasma     113 

for  human  seminal  plasma,  is  probably  due  to  this  mucoid  sub- 
stance (Wislocki,  1950). 

The  proteoses  in  trichloroacetic  acid  extracts  from  semen,  which 
one  encounters  not  only  in  man,  but  also  in  the  ram,  bull,  boar, 
and  other  species,  occasionally  interfere  with  chemical  analyses  of 
certain  non-protein  constituents.  To  overcome  this  difficulty,  it  is 
advisable  to  replace  trichloroacetic  acid  with  other  deproteinizing 
agents  such  as  zinc  hydroxide,  tungstic  acid,  phosphotungstic  acid 
or  ethanol.  Another  source  of  trouble  encountered  in  analytical 
work  with  semen  and  due  to  the  mucinous  substance  in  seminal 
plasma,  human  in  particular,  is  that  on  centrifugation  the  mucus 
has  a  tendency  to  form  a  stringy  mass  which  firmly  adheres  to  the 
sperm  cells.  Caution  must  therefore  be  exercised  in  attributing  to 
spermatozoa  as  such,  analytical  results  obtained  with  centrifuged 
human  semen. 

Several  assays  of  free  amino  acids  in  mammalian  seminal  plasma 
have  been  carried  out,  mostly  however,  by  chromatographic  or 
microbiological  methods,  and  not  by  chemical  isolation.  A  notable 
exception  is  the  work  of  Wagner- Jauregg  (1941)  who  isolated 
crystalline  tyrosine  from  an  ethanolic  extract  of  human  semen.  The 
following  free  amino  acids  were  found  to  occur  in  the  seminal  plasma 
of  man,  glycine,  threonine,  alanine,  valine,  leucine,  isoleucine, 
cystine,  proline,  tyrosine,  phenylalanine,  lysine,  arginine,  aspartic 
acid  and  glutamic  acid  (Jacobsson,  1950;  Lundquist,  1952).  In 
bovine  seminal  plasma  serine,  glycine,  alanine,  aspartic  acid  and 
glutamic  acid  were  found  (Gassner  and  Hopwood,  1952),  and  a 
similar  pattern  was  also  observed  in  the  seminal  vesicle  secretion 
and  in  the  ampullar  fluid,  the  latter  containing  in  addition  a  trace  of 
tyrosine.  According  to  Gassner,  the  free  amino  acids  in  bull  seminal 
plasma  disappear  after  castration,  like  seminal  fructose  and  citric 
acid,  but  their  content  is  not  restored  by  testosterone  administra- 
tion; furthermore,  vasectomy  alone,  which  is  without  effect  on  the 
content  of  fructose,  causes  a  disappearance  of  amino  acids  from  bull 
seminal  plasma. 

Free  amino  acids  occur  also  in  fish  semen;  as  long  ago  as  1923, 
leucine,  lysine  and  alanine  have  been  isolated  in  pure  form  from 
protein-free  extracts  of  herring  testicles  (Steudel  and  Suzuki,  1923). 

There  are  indications  that  the  amino  acids  and  proteoses  present 

114  The  Biochemistry  of  Semen 

in  the  seminal  plasma  may  be  of  some  importance  to  the  sperma- 
tozoa. It  may  be  recalled  that  excessive  dilution  of  semen  exerts  a 
deleterious  effect  on  spermatozoa  and  that  this  can  be  counteracted, 
partly  at  least,  by  the  inclusion  in  the  diluting  media  of  amino  acids 
such  as  glycine,  alanine,  valine,  leucine,  lysine,  and  glutamic  acid 
(p.  76).  The  beneficial  action  of  these  amino  acids  is  believed  to 
depend  primarily  on  their  metal-binding  capacity  (Tyler  and  Roth- 
schild, 1951).  Several  other  effects  of  amino  acids  have  been  observed 
with  the  sperm  of  lower  animals.  Giese  and  Wells  (1952)  found 
that  glycine  (005m)  protected  the  spermatozoa  of  Strongylocentrotus 
piirpwatus  from  the  detrimental  effect  of  light.  Metz  and  Donovan 
(1950)  demonstrated  that  in  the  starfish  certain  amino  acids  promote 
the  agglutination  of  spermatozoa  by  egg-water  of  this  species;  in 
the  absence  of  these  amino  acids  agglutination  does  not  take  place. 

Fibrinolysin  and fibrinogenase 

The  seminal  proteoses  and  amino  acids  are  presumably  the  pro- 
ducts of  proteolytic  activity  which  in  the  seminal  plasma  is  derived 
mainly  from  the  prostatic  secretion,  but  partly  also  from  the 
seminal  vesicle  fluid.  The  two  powerful  proteolytic  agents  of  the 
prostatic  secretion  are  'fibrinolysin'  and  'fibrinogenase'  (see  also 
pp.  17  and  29). 

The  coagulation  of  human  semen  is  followed  by  liquefaction,  a 
process  which  is  catalysed  by  a  proteolytic  agent  present  in  the 
prostatic  secretion.  Its  discoverers,  Huggins  and  Neal  (1942)  named 
it  fibrinolysin  because  of  its  ability  to  digest  blood  fibrin,  and  its 
resemblance  to  the  fibrinolytic  agent  in  haemolytic  streptococci 
(Tillet  and  Garner,  1933).  However,  the  fibrinolytic  system  present 
in  blood  has  now  been  resolved  into  several  distinct  components, 
whereas  the  streptococcal  fibrinolysin  has  been  defined  as  a  kinase, 
i.e.  an  activator  of  the  fibrinolytic  enzyme  preformed  in  the  blood. 
Consequently,  the  name  'fibrinolysin'  has  been  abandoned  with 
reference  to  the  streptococcal  agent  in  favour  of  'streptokinase'. 
Furthermore,  it  proved  impossible  to  replace  streptokinase  by  pro- 
static fibrinolysin  as  an  activator  of  the  blood  enzyme  (Oettle, 

The  fibrinolytic  activity  can  be  assayed  in  human  semen  by  the 
method  of  Harvey  (1949),  which  consists  in  mixing  a  constant  volume 

Protein  Constituents  and  Enzymes  of  Seminal  Plasma     1 1 5 

of  oxalated  blood  plasma  with  varying  volumes  of  semen,  inducing 
clotting  by  the  addition  of  1-5%  calcium  chloride,  and  noting  the 
time  required  for  the  clot  to  liquefy.  Owing  to  the  inhibitory  effect 
of  blood  plasma  on  fibrinolysis,  the  plasma  must  not  constitute 
more  than  one-tenth  of  the  reacting  system.  Harvey  states  that  the 
degree  of  fibrinolytic  activity  in  semen  varies  with  the  individual 
but  she  found  no  correlation  between  this  activity  and  either  the 
volume  of  ejaculates  or  any  characteristics  of  spermatozoa.  Simi- 
larly, there  was  no  positive  relationship  between  the  lysin  content 
and  semen  viscosity.  However,  specimens  which  were  exceptionally 
viscous,  usually  also  had  low  fibrinolytic  power. 

The  precise  nature  of  seminal  fibrinolysin  and  its  relation  to 
plasmin,  the  fibrin-splitting  agent  of  the  blood,  will  not  be  known 
until  the  enzyme  has  been  purified.  Moreover,  experiments  by  Kaulla 
and  Shettles  (1953)  indicate  that  in  addition  to  the  plasmin-like 
enzyme  proper,  the  human  seminal  plasma  contains  at  least  three 
other  agents,  (i)  fibrinolysokinase,  an  activator  of  blood  profibrinoly- 
sin,  (ii)  a  small  amount  of  profibrinolysin  itself,  i.e.  of  material 
which  can  be  activated  by  streptokinase,  and  (iii)  antifibrinolysiny 
an  inhibitor  of  plasmin,  which,  however,  is  present  in  a  much  lower 
concentration  in  the  seminal  plasma  than  in  blood  serum. 

Fibrinogenase  is  the  name  given  by  Huggins  and  Neal  (1942) 
to  the  proteolytic  agent,  highly  active  in  canine  prostatic  secretion, 
but  less  so  in  human  prostatic  fluid,  which  destroys  blood  plasma 
fibrinogen.  Huggins  and  his  co-workers  (1942,  1943)  also  share  the 
credit  for  having  recognized  the  similarity  between  certain  other 
proteolytic  properties  of  the  prostatic  secretion  and  those  of  pan- 
creatic trypsin.  More  recently,  the  'tryptic'  enzyme  of  human  semen 
has  been  partially  purified  by  Lundquist  (1952),  who  defined  as  a 
unit  the  amount  of  enzyme  which  in  1  hr.,  at  pH  7-6,  and  37°, 
liberates  from  added  casein  a  quantity  of  chromogen  corresponding 
to  01  mg.  free  tyrosin;  he  achieved  an  activity  of  about  1  unit  per 
mg.  of  protein-nitrogen,  i.e.  an  approximately  tenfold  purification. 
The  purified  enzyme  was  active  also  towards  haemoglobin  and  both 
human  and  bovine  blood  plasma  fibrinogen.  In  an  attempt  to 
purify  from  human  semen  the  natural  substrate  for  the  proteolytic 
activity,  Lundquist  obtained  a  protein  fraction,  'seminal  fibrin', 
which  was  readily  digested  by  enzyme  preparations  both  from 

1 1 6  The  Biochemistry  of  Semen 

semen  and  the  prostate  gland,  and  yielded  the  same  amino  acids 
which  appear  normally  in  human  semen. 


Apart  from  the  above  mentioned  protease  which  acts  optimally 
at  pH  7-6,  two  more  proteolytic  enzymes  have  been  found  in 
human  semen  by  Lundquist  and  his  co-workers  (1951,  1952,  1953). 
One  is  an  amino  peptidase  which  hydrolyses  leucine  amide,  glycyl- 
glycine,  triglycine  and  glycyl- leucine,  with  an  optimum  around  pH 
7-5.  The  other  has  been  identified  as  pepsinogen.  According  to 
Lundquist  and  Seedorff  (1952),  the  activity  of  pepsinogen  in  semen 
corresponds  to  2  //g.  pepsin /ml.,  which  is  of  the  same  order  of 
magnitude  as  that  found  in  gastric  juice.  But  unlike  the  trypsin- 
like  enzyme,  seminal  pepsinogen  seems  to  originate  in  the  seminal 
vesicles  and  not  in  the  prostate.  Its  specific  function  is  not  fully 
understood  as  it  is  difficult  to  envisage  in  semen  the  high  hydrogen 
ion  concentration  required  for  the  conversion  of  pepsinogen  to 

Ammonia  formation 

A  phenomenon  probably  associated  with  the  enzymic  degrada- 
tion of  proteins,  is  the  progressive  accumulation  of  free  ammonia 
which  takes  place  in  whole  semen  and  in  seminal  plasma,  on  anaero- 
bic as  well  as  aerobic  incubation.  This  has  been  observed  in  several 
species  (Shergin,  1933;  Mann,  1945^).  In  ram  semen  for  example, 
the  content  of  free  ammonia  (estimated  by  vacuum-steam  distilla- 
tion in  the  Parnas-Heller  apparatus)  was  found  to  increase  from 
1-3  mg.  NH3-N/IOO  ml.  in  fresh  semen  to  9-7  mg.  NH3-N/IOO  ml., 
after  7  hr.  incubation  at  37°,  under  sterile  conditions  (Mann, 
\9A5a).  Ammonia  formed  in  semen  is  in  considerable  excess  of  the 
amount  which  could  be  derived  from  adenyl  derivatives;  the  total 
adenine  amino-A'^  content  of  the  semen  as  assessed  enzymically 
with  heart  muscle  deaminase  being  only  about  4  mg./lOO  ml.  in 
fresh,  and  3  mg./lOO  ml.  in  incubated,  semen.  It  is  also  unlikely  to 
originate  from  urea,  since  the  content  of  urea  in  semen  is  not  sig- 
nificantly affected  by  incubation. 

Protein  Constituents  and  Enzymes  of  Seminal  Plasma     1 1 7 

Amino  acid  oxidase 

In  addition  to  sugars  and  fatty  acids,  spermatozoa  are  capable 
of  oxidizing  a  number  of  amines  and  amino  acids.  The  oxidative 
deamination  of  amino  acids  by  bull  spermatozoa  has  been  the  subject 
of  a  study  by  Tosicand  Walton  (1945;  1946«,  b,  1950).  The  starting 
point  of  this  study  has  been  the  observation  that  the  addition  of 
egg-yolk  to  bull  sperm  causes  an  increased  oxygen  uptake,  which, 
however,  gradually  declines  in  about  an  hour's  time.  Egg-yolk 
fractionation  led  to  a  dialysable,  nitrogen-containing  fraction  which 
was  oxidized  by  spermatozoa.  The  oxidation  was  accompanied  by 
accumulation  of  ammonia  and  formation  of  hydrogen  peroxide. 
Evidence  was  obtained  which  pointed  to  peroxide  being  responsible 
for  the  gradual  decline  in  the  oxygen  uptake  by  spermatozoa. 

In  the  course  of  their  study,  Tosic  and  Walton  examined  several 
pure  amino  acids  and  found  that  spermatozoa  oxidize  three  natur- 
ally occurring  amino  acids,  namely  L-tyrosine,  L-phenylalanine  and 
L-tryptophan.  According  to  Tosic  (1947,  1951),  the  hydrogen- 
peroxide-forming  aerobic  process  in  bull  semen  is  an  oxidative 
deamination  catalysed  by  the  L-amino  acid  oxidase  of  spermatozoa, 
which  differs  from  the  analogous  enzyme  of  other  animal  tissues 
by  having  its  range  of  activity  restricted  to  only  three  aromatic 
amino  acids;  the  activity  of  the  enzyme  can  be  expressed  by  the 

R  CH2CHCOOH+O2+H2O  — >  R  CH,  C  COOH+H2O2+NH3 

I  "II 

NH2  O 

Seminal  phosphatases 

Semen  owes  its  powerful  phosphatase  activity  mainly  to  the 
seminal  plasma  which  carries  several  different  dephosphorylating 
enzymes  derived  from  the  male  accessory  organs  of  reproduction. 
Among  the  most  active  and  best  known  enzymes  in  this  group  are  the 
so-called  'acid  phosphatase'  and  'alkaline  phosphatase'.  In  addition 
to  these  two  phosphomonoesterases,  the  seminal  plasma  contains 
'5-nucleotidase',  a  pyrophosphatase,  and  several  adenosinetri- 

In   early   studies   on  phosphatases,  the   substrates  commonly 

118  The  Biochemistry  of  Semen 

used  were  a-  and  /3-phosphoglycerol,  and  phosphohexoses,  chiefly 
6-phosphoglucose  and  6-phosphofructose,  but  also  1  :  6-diphospho- 
fructose.  More  recently,  however,  other  organic  phosphoric  acid 
derivatives  came  into  use,  including  various  nucleotides  and  inter- 
mediary phosphorylated  compounds  of  glycolysis,  as  well  as  two 
synthetic  substances:  phenylphosphate  (King  and  Armstrong,  1934) 
and  phenolphthaleine  phosphate  (King,  1943;  Huggins  and  Talalay, 
1945).  The  introduction  of  histochemical  techniques  marked  another 
important  development  in  studies  on  phosphatases  (Gomori,  1939, 
\94la,  b,  1953).  The  histochemical  investigations  have  thrown  much 
light  upon  the  pattern  of  phosphatase  distribution  in  the  male 
accessory  organs  and  have  helped  to  establish  the  existence  of 
'secretory'  phosphatases,  localized  in  the  secretory  epithelia  and 
secretions  of  accessory  glands,  as  distinct  from  the  'stromal'  phos- 
phatases which  are  present  only  in  the  stroma  (Dempsey,  1948; 
Bern,  1949;  RoUinson,  1954). 

''Acid'  and  'alkaline'  phosphatase 

An  observation  that  the  phosphatase  activity  of  male  urine  is 
usually  higher  than  in  women,  led  Kutscher  and  Wolbergs  (1935) 
to  examine  the  phosphatase  in  semen  and  in  the  prostate  gland. 
They  soon  found  that  semen  and  prostate  are  among  the  richest 
sources  of  acid  phosphatase  in  the  human  body,  the  enzyme  being 
optimally  active  at  pH  5-6,  equally  well  towards  a-  and  ^S-phospho- 
glycerol,  but  largely  inactive  towards  diphosphofructose  and  pyro- 
phosphate (Kutscher  and  co-workers,  1936,  1938).  Subsequent 
investigations  confirmed  and  extended  these  findings;  the  demon- 
stration by  Scott  and  Huggins  (1942)  that,  while  the  voided  urine 
of  man  is  rich  in  the  enzyme,  urine  collected  directly  from  the  renal 
pelvis  shows  only  little  enzymic  activity,  was  a  convincing  proof 
that  the  content  of  acid  phosphatase  in  normally  voided  male  urine 
is  due  largely  to  the  admixture  of  prostatic  secretion. 

Acid  phosphatase  is  an  important  secondary  male  sex  charac- 
teristic. Investigations  by  Gutman  and  Gutman  (1938Z))  have  shown 
that  the  level  of  the  enzyme  in  the  human  prostate  is  low  in  child- 
hood but  increases  rapidly  at  puberty;  thus  the  activity,  expressed  in 
King- Armstrong  units  per  gram  prostate  tissue,  was  H  units  at  four 
years  of  age,  73  units  at  puberty,  and  522  to  2284  units  in  adult  men. 

Protein  Constituents  and  Enzymes  of  Seminal  Plasma     119 

A  similar  relation  to  age  was  observed  in  monkeys  and  dogs;  in 
both  these  species  administration  of  androgenic  hormones  to  imma- 
ture males  stimulates  considerably  the  output  of  the  enzyme  from 
the  prostate  gland  (Gutman  and  Gutman,  1939;  Huggins  and 
Russell,  1946).  A  certain  correlation  appears  to  exist  in  adult  men 
between  the  level  of  acid  phosphatase  in  semen  and  androgenic 
activity  (Gutman  and  Gutman,  1940;  Gutman,  1942;  Engberg, 
Anderson,  Sury  and  Raft,  1947).  However,  like  other  constituents 
of  semen,  the  level  of  prostatic  phosphatase  activity  varies  from  one 
species  to  another,  as  well  as  between  individuals  within  the  same 

Under  physiological  conditions,  acid  phosphatase  does  not  pass 
from  the  prostate  into  the  blood  stream.  However,  significant 
amounts  of  it  appear  in  the  blood  plasma  as  a  result  of  malignant 
growth  in  the  prostate  and  metastases  of  prostatic  cancer  in  the 
bones;  injections  of  androgen  still  further  increase  the  level  of 
enzyme  in  blood  plasma,  whereas  castration  or  treatment  with 
oestrogens  lead  to  a  spectacular  decrease.  The  determination  of 
prostatic  phosphatase  activity  in  blood  has  been  utilized  as  a  valuable 
diagnostic  aid  in  prostatic  carcinoma  and  in  the  course  of  clinical 
treatment  (Gutman  and  Gutman,  1938a;  Huggins,  Scott  and  Hodges, 
1941;  Watkinson,  Delory,  King  and  Haddow,  1944). 

An  important  addition  to  our  knowledge  of  the  physiological 
functions  of  acid  phosphatase  in  seminal  plasma,  has  been  the  dis- 
covery made  by  Lundquist  (1946)  that  freshly  ejaculated  human 
semen  contains  phosphorylcholine  which  on  ejaculation  is  rapidly 
dephosphorylated  by  the  acid  phosphatase  to  free  choline  and 
orthophosphate.  This  phenomenon  is  more  fully  discussed  else- 
where (p.  170).  But  there  is  evidence  that  apart  from  the  acid  phos- 
phatase which  acts  on  phosphorylcholine  optimally  at  pH  6-3  (in 
acetate  buffer),  human  seminal  plasma  contains  another  phosphatase 
which  acts  on  the  same  substrate  at  a  higher  pH  (Hudson  and  Butler, 
1950).  Of  considerable  interest  is  also  the  finding  that  the  acid 
seminal  phosphatase  exhibits  in  vitro  a  distinct  transferase  activity 
(Green  and  Meyerhof,  1952);  partially  purified  acid  phosphatase 
from  the  human  prostate  has  been  shown  to  catalyse  at  pH  5-5 
the  transfer  of  phosphate  from  both  ^S-phosphoglycerol  and  phospho- 
creatine  to  glucose;  the  product  in  each  case  was  6-phosphoglucose 


The  Biochemistry  of  Semen 

(Morton,  1953).  Methods  for  the  purification  of  acid  phosphatase 
from  human  prostate  glands  and  semen  have  been  described  by 
London  and  Hudson  (1953)  and  Boman  (1954). 

Alkaline  phosphatase,  like  the  'acid'  enzyme,  is  widely  distributed 
in  male  accessory  organs  but  its  localization  in  cells  and  concentra- 
tion in  accessory  gland  secretions  is  different.  Human  semen  with 
its  conspicuously  high  level  of  acid  phosphatase,  has  a  low  concen- 
tration of  the  alkaline  phosphatase.  Bull  semen  on  the  other  hand, 
has  only  slight  acid  phosphatase  activity  but  contains  more  of  the 
alkaline  phosphatase  (Haq  and  Mullen,  1948;  Reid,  Ward  and 
Salsbury,  1948^).  This  difference  between  the  human  and  bovine 
semen  is  not  altogether  unexpected,  since  the  bulk  of  bull  seminal 
plasma  is  derived  not  from  the  prostate  but  from  the  seminal 
vesicles.  In  the  rat  both  phosphatases  are  of  low  activity;  with  the 
exception  of  the  ventral  prostate  which  may  contain  up  to  20  units 
of  alkaline  phosphatase  per  g.  tissue,  the  level  of  either  enzyme 
seldom  exceeds  4  units  per  g.  in  any  one  of  the  other  accessory 
organs.  After  castration  the  activity  of  both  these  enzymes  diminishes 
first  in  the  rat  seminal  vesicles,  and  a  little  later  in  the  prostate;  but 
the  percentage  decrease  of  enzymic  activity  and  of  organ  weight  is 
roughly  equal  (Huggins  and  Webster,  1948;  Stafford,  Rubinstein 
and  Meyer,  1949). 

Table  17.  Phosphatase  activity  of  ram  seminal  plasma  on 
phosphohexoses  (Mann  and  Lutwak-Mann,  19516) 

(The  liberation  of  sugars  was  examined  by  incubating  5  mg.  substrate 
(Na  salt)  with  1  ml.  dialysed  seminal  plasma  at  pH  7  or  0-2  ml.  dialysed 
seminal  plasma  at  pH  9,  for  1  hr.,  37°,  in  the  presence  of  OOOSM-MgCla. 
The  sugars  were  determined  after  deproteinization  with  ZnS04  and 
Ba(OH)2;  glucose  was  estimated  by  means  of  glucose  oxidase  (Mann, 
1944;  \9A6b).) 

Fructose  (%) 

Glucose  (%) 

Inorg.  phosphate  (%) 





pH=7          pH=9 






67             93 






94            100 






97            100 






94            100 

1  :  6-Diphosphofructose 





20              95 






60            100 

Protein  Constituents  and  Enzymes  of  Seminal  Plasma     121 

Alkaline  phosphatase  has  an  optimum  at  about  pH  9,  and  is 
capable  of  hydrolysing  among  others,  1-phosphofructose,  6-phos- 
phofructose  and  1  :  6-diphosphofructose.  Our  researches  indicate 
that  this  activity  may  represent  an  essential  step  in  the  process  of 
fructose  formation  and  secretion  by  the  accessory  organs  (Mann 
and  Lutwak-Mann,  1951«,  b)  (see  also  p.  150).  Ram  seminal  plasma 
is  particularly  rich  in  alkaline  phosphatase  which,  though  it  acts 
optimally  at  pH  9,  also  shows  appreciable  activity  towards  phos- 
phohexoses  at  pH  7.  This  can  be  seen  from  Table  17  which  gives 
rates  of  dephosphorylation  for  various  compounds.  In  the  case  of 
6-phosphofructose  and  1  :  6-diphosphofructose,  some  glucose  is 
formed  in  addition  to  fructose,  owing  to  the  presence  of  phospho- 
hexose  isomerase  in  the  seminal  plasma,  as  a  result  of  which  a  part 
of  6-phosphofructose  is  converted  to  6-phosphoglucose  before 
dephosphorylation.  Supporting  evidence  for  the  conclusion  that 
monophosphofructose  rather  than  diphosphofructose,  is  the  sub- 
strate immediately  responsible  for  the  liberation  of  seminal  fructose, 
has  been  provided  by  Bouchilloux  and  Menager  (1952)  who  found 
that  the  semen  of  both  ram  and  bull  contains  two  phosphomono- 
esterases,  with  pH  optima  at  9-4  and  4-8,  respectively,  but  that  it 
lacks  a  specific  fructosediphosphatase. 


This  enzyme  was  discovered  in  human  semen  by  Reis  (1937, 
1938,  1940)  and  shown  to  dephosphorylate  muscle  adenylic  acid 
(adenosine-5 '-phosphoric  acid)  and  inosinic  acid  (inosine-5'-phos- 
phoric  acid)  but  not  adenosine-3 '-phosphoric  acid  or  adenosine 
triphosphoric  acid.  Bull  seminal  plasma  is  particularly  rich  in  5- 
nucleotidase,  the  seminal  vesicles  being  the  main  source  of  the 
enzyme  (Mann,  1945a,  1947).  Bull  seminal  plasma  or  the  vesicular 
secretion  itself,  act  several  hundred  times  more  efficiently  on  muscle 
adenylic  acid  than  on  /S-phosphoglycerol;  from  160  ^g.  P  added  as 
sodium  adenylate  to  0001  ml.  bull  seminal  plasma,  up  to  140  /^g. 
P  are  liberated  as  orthophosphate  during  1  hour's  incubation  at  37°. 

The  5-nucleotidase  of  bull  seminal  plasma  has  been  purified  about 
fifty-fold  by  Heppel  and  Hilmoe  (195 la).  The  purified  enzyme 
has  a  pH  optimum  at  8-5,  and  its  activity  is  enhanced  by  the  addi- 
tion of  magnesium  ions  but  inhibited  by  fluoride  (00  1m)  and  by 

122  The  Biochemistry  of  Semen 

borate  buffer  (0-08m)  to  the  extent  of  73  and  85%  respectively.  On 
the  basis  of  tests  with  numerous  phosphorylated  compounds,  it 
may  be  safely  concluded  that  5-nucleotidase  is  an  enzyme  which  acts 
specifically  on  substrates  containing  the  ribose-5-phosphate  moiety. 
The  purified  enzyme  splits  rapidly  ribose-5-phosphate  but  not 
ribose-3-phosphate;  it  is  active  towards  adenosine-5 '-phosphate, 
inosine- 5 '-phosphate,  uridine-5'-phosphate  and  cytidine-5'-phos- 
phate  but  inactive  towards  both  adenosine-3 '-phosphate  and 
adenosine-2'-phosphate.  It  also  dephosphorylates  nicotinamide 
mononucleotide  (nicotinamide  ribose-5'-phosphate);  this  inciden- 
tally explains  an  early  observation  of  ours,  that  bull  seminal  plasma 
decomposes  cozymase,  with  slow  liberation  of  inorganic  phosphate. 


Bull  seminal  plasma  contains  an  enzyme  which  hydrolyses  in- 
organic pyrophosphate  to  orthophosphate  but  differs  from  the 
pyrophosphatase  of  yeast.  Seminal  pyrophosphatase  can  exert  its 
maximal  activity  in  the  absence  of  magnesium  ions  and  is  not 
inhibited  by  increased  substrate  concentration;  it  has  a  sharp 
optimum  at  pH  8-6  (Heppel  and  Hilmoe,  \95\b), 

Enzymic  hydrolysis  of  adenosine  triphosphate 

In  addition  to  adenosine-triphosphatase  (ATP-ase)  in  the  sperma- 
tozoa there  are  also  ATP-splitting  enzymes  in  the  seminal  plasma 
(Mann,  1945o;  MacLeod  and  Summerson,  1946).  When  adenosine 
triphosphate  is  acted  upon  by  bull  or  human  seminal  plasma,  all 
three  phosphate  groups  are  set  free  as  orthophosphate.  The  mechan- 
ism of  this  reaction  has  been  investigated  by  Heppel  and  Hilmoe 
(1953)  who  by  fractionation  procedures  obtained  three  distinct 
ATP-ases,  none  of  them,  however,  completely  free  from  5-nucleo- 

One  of  the  enzymes,  named  the  'pyrophosphate-forming  ATP- 
ase',  catalyses  the  reaction 

Adenosine  triphosphate +H2O  — >- 


It  is  relatively  heat-stable,  has  a  pH  optimum  at  8 -4-8 -6  and 
requires  neither  calcium  nor  magnesium  ions  for  activation.  The 

Protein  Constituents  and  Enzymes  of  Seminal  Plasma     123 

remaining  two  ATP-ases,  designated  respectively  as  'acid'  and 
'alkaline',  produce  orthophosphate;  the  'acid'  ATP-ase  has  a  pH 
optimum  at  5-7-60,  requires  magnesium,  is  inhibited  by  calcium, 
and  can  be  inactivated  completely  by  heating  for  20  min.  at  60°;  it 
does  not  act  on  i5-phosphoglycerol;  the  'alkaline'  ATP-ase  is  more 
heat-resistant,  has  a  pH  optimum  at  8 -4-8 -8,  and  is  stimulated  by 
calcium  and  also  by  magnesium.  Both  these  enzymes  are  active  not 
only  tov^ards  adenosine  triphosphate  but  adenosine  diphosphate 
as  v^ell. 

Bull  seminal  plasma  contains  in  1  ml.  about  80  units  of  acid 
ATP-ase,  130  units  of  alkaline  ATP-ase,  40  units  of  the  pyrophos- 
phate-forming  ATP-ase,  and  2900  units  of  5-nucleotidase.  It  re- 
mains for  future  studies  to  define  the  physiological  significance 
of  all  these  enzymes,  particularly  the  5-nucleotidase  which  is  so 
characteristic  of  semen.  Possibly,  there  is  some  link  between  them 
and  other  nucleolytic  enzymes  and  they  may  well  play  a  role  in  the 
metabolism  of  purine  compounds  in  semen  and  reproductive  organs. 
The  occurrence  of  nucleases  in  human  and  sea-urchin  semen 
(Zamenhof,  Shettles  and  Chargaff,  1950;  Mazia,  1949),  the  cozy- 
mase-destroying  activity  of  bull  seminal  plasma  (Mann,  1945a),  the 
interesting  findings  on  the  presence  of  uric  acid  in  bull  semen 
(Barron  and  Haq,  1948;  Leone,  1952),  and  the  more  recent  demon- 
stration of  xanthine  oxidase  in  the  bull  vesicular  secretion  (Leone, 
1953),  are  but  a  few  examples  of  problems  in  this  field,  which  await 
further  and  more  detailed  study. 


Lipids  and  their  Role  in  the 
Metabolism  of  Semen 

Lipids  in  spermatozoa.  The  lipid  capsule.  Acetal  phospholipids  or  plas- 
malogens.  Role  of  lipids  in  sperm  metabolism.  Lipids  in  the  seminal  plasma 
and  male  accessory  gland  secretions.  'Lipid  bodies'  and  prostatic  calculi. 

Lipids  in  spermatozoa 

The  first  systematic  analysis  of  lipids  in  spermatozoa  was  carried 
out  by  Miescher  (1878,  1897)  who  also  proved  that  the  lipids  are 
concentrated  chiefly  in  the  sperm-tails.  His  analytical  results  showed 
that  the  ether-extractable  material  obtained  from  salmon  sperma- 
tozoa is  composed  of  about  50%  lecithin,  14%  cholesterol  and 
35%  fat,  and  that  by  far  the  greatest  part  of  this  material  is  derived 
from  the  sperm-tails  where  lecithin  accounts  for  31-83%,  fats  and 
cholesterol  for  26-27%,  and  protein  for  the  remaining  41 -90%  of  the 
organic  contents.  This  led  Miescher  to  conclude  that  the  sperm- 
tails  resemble  in  their  composition  the  grey  matter  of  the  nervous 
system,  and  in  a  letter  to  W.  Hiss  he  wrote:  'The  more  I  deal  with 
the  tails,  the  more  probable  it  appears  to  me  that  we  have  before 
us  essentially  the  chemical  type  of  the  non-medullated  nerves,  that 
is  the  axis  cylmders.'  Subsequent  investigations  by  Mathews  (1897) 
and  Sano  (1922)  on  the  sperm  of  herring,  salmon,  porgy  and  cod- 
fish, confirmed  the  presence  of  lecithin  and  revealed  at  the  same 
time  the  presence  of  small  quantities  of  certain  other  lipids,  includ- 
ing cephalin  and  sphingomyelin. 

H2C — O — CORunsat. 

HC— O— CORsat.  Lecithin 


H2C— O— P— O— CH2-CH2 

I         I 

O-  +N(CH3)3 


Lipids  and  their  Role  in  the  Metabolism  of  Semen     125 

When  air-dried  salmon  roe  is  ground  and  extracted  with  pentane 
in  a  Soxhlet  apparatus,  a  yellow  oil  is  obtained  which  is  practically 
free  from  phospholipids;  it  requires  further  treatment  with  ethanol 
or  methanol  for  the  phospholipids  to  be  extracted  from  the  sperm. 
In  this  way,  for  example,  12-5%  glyceride  in  the  oily  fraction,  and 
6-2%  phospholipid  in  the  alcoholic  fraction,  was  obtained  from  the 
roe  of  the  sockeye  salmon,  Oncorhynchus  nerka  (Halpern,  1945).  On 
the  basis  of  this  observation,  it  has  been  suggested  that  the  phos- 
pholipids occur  in  the  spermatozoa  in  a  firmly  bound  state,  pre- 
sumably in  the  form  of  lipoproteins.  A  substantial  portion  of  the 
unsaponifiable  material  extracted  from  fish  sperm  by  fat  solvents 
consists  of  cholesterol  which  accounts,  on  the  average,  for  2-2% 
of  dried  fish  spermatozoa  (Schmidt-Nielsen  and  Sundsvold,  1943). 

The  high  content  of  lipids  in  spermatozoa  is  equally  characteristic 
for  fishes  as  for  other  animals.  Sea-urchin  spermatozoa  are  well 
known  to  contain  a  large  reserve  of  lipid  material,  shown  by  Mathews 
(1897)  to  include  both  lecithin  and  neutral  fat.  The  content  of  phos- 
pholipids in  the  sperm  of  Echinus  esculent  us  is  about  5-5%  of  the 
dry  weight  of  spermatozoa  (Rothschild  and  Cleland,  1952).  The 
seminal  lipids  of  E.  esculentus  have  been  analysed  more  recently  by 
Cardin  and  Meara  (1953).  The  material  obtained  by  extraction  of 
1-2 1.  of  semen  with  acetone  and  light  petroleum,  consisted  of  13-6% 
neutral  fat,  32-9%  free  fatty  acids,  260%  phospholipids,  9-2% 
sterols  and  18-3%  of  other  unsaponifiable  matter.  The  component 
fatty  acids  of  the  non-phospholipid  fraction  included  a  low  propor- 
tion (10-1%)  of  saturated  acids  and  a  high  proportion  of  unsatur- 
ated acids  with  18C  (30-4%)  20C  (451%)  and  22C  (12-3%).  The 
phospholipid  fraction  had  a  ratio  of  N  :  P=l-4  :  1,  and  must  have 
therefore,  consisted  of  a  mixture  of  monoaminophosphatides  and 
diaminophosphatides . 

Early  analyses  of  lipids  in  bovine  epididymal  sperm  were  carried 
out  by  Koelliker  (1856)  who  found  that  over  12%  of  the  dried 
material  is  ether-extractable.  About  half  of  this  content  was  later 
shown  by  Miescher  (1878)  to  consist  of  lecithin.  In  a  study  of  the 
lipid  content  of  bull  sperm,  Zittle  and  O'Dell  (1941«)  have  extracted 
washed  epididymal  spermatozoa  successively  with  ethanol,  ether, 
acetone  and  petroleum  ether,  and  found  13%  of  lipid  material;  when 
the  procedure  was  repeated  with  spermatozoa  disintegrated  by  sonic 

126  The  Biochemistry  of  Semen 

treatment,  more  lipid  was  found  in  the  tails  (23%)  than  in  the  mid- 
pieces  (6%)  or  heads  (7%). 

The  lipid  capsule 

It  is  probable  that  the  high  lipid  content  of  spermatozoa  is  due 
largely  to  the  lipid  'sheath'  or  'capsule'  which  encloses  the  sperm 
cell.  So  it  would  seem  at  any  rate,  from  histochemical  studies, 
including  the  extensive  investigation  of  Popa  and  Marza  (1931)  who 
described  the  so-called  manteau  Upidique  in  the  spermatozoon  of 
man,  dog,  bull,  ram,  boar,  rabbit,  guinea-pig  and  cock.  The  lipid 
capsule  is  presumably  of  importance  to  the  spermatozoa  in  their 
function,  perhaps  to  ward  off  the  effects  of  the  acid  vaginal  milieu  as 
has  been  suggested  by  Redenz  (1924).  It  appears  to  consist  largely 
of  a  lipoprotein  complex  which  is  fairly  soluble  in  aqueous  solvents. 
In  the  case  of  mammalian  spermatozoa,  this  complex  has  been 
extracted  with  a  01  4m  solution  of  sodium  chloride  at  pH  9  (Dallam 
and  Thomas,  1952).  In  the  middle-piece  lipids  were  shown  to  be 
associated  with  the  'spiral  body'  which  surrounds  the  axial  proto- 
plasmic thread,  and  is  derived  from  the  mitochondria  of  the  sperma- 
tids (Wislocki,  1950;  Brown,  1952). 

Acetal phospholipids  or  plasmalogens 

An  interesting  feature  of  the  sperm  cell  is  a  characteristically  high 
content  of  acetal  phospholipids  or  plasmalogens.  Feulgen  and 
Rosenbeck  (1924)  while  studying  the  'nucleal'  reaction  of  human 
spermatozoa,  noted  that  when  fresh  smears  of  human  semen  were 
treated  with  Schiff's  fuchsin-sulphurous  acid  reagent,  the  middle- 
pieces  and  tails,  though  devoid  ojf  nuclear  material,  stained  strongly. 
This  observation  was  followed  by  a  demonstration  that  cells  in 
general  contain  in  their  protoplasm  some  material  which  stains 
diffusely  with  Schiff's  reagent,  but  differs  from  nucleoproteins  by  its 
solubility  in  ethanol.  The  name  'plasmal'  was  bestowed  upon  this 
material,  which  was  shown  in  subsequent  investigations  by  Feulgen 
and  his  co-workers  to  arise  from  'plasmalogen',  a  group  of  peculiar 
phospholipids  widely  distributed  in  tissues,  and  distinguished  by  the 
presence  of  higher  fatty  aldehydes  in  place  of  the  usual  fatty  acids. 
The  plasmalogen  isolated  by  Feulgen  and  Bersin  (1939)  from  beef 
muscle  was  identified  as  an  acetal  of  glycerylphosphorylcolamine. 

Lipids  and  their  Role  in  the  Metabolism  of  Semen     127 

The  two  principal  fatty  aldehydes  in  plasmalogens  are  palmitic  and 
stearic  aldehydes  but  other  fatty  aldehydes  were  also  reported 
(Feulgen,  Boguth  and  Andresen,  1951).  Crystalline  acetal  phos- 
pholipids were  prepared  from  beef  brain,  and  shown  to  belong  to 
the  a-series  (Thannhauser,  Boncoddo  and  Schmidt,  1951). 

H.C— O 


HaC — O  Acetal  a-phospholipid 

I  O 

I  II 

H,C— O— P— O— CH, 

I  1 

OH       NH2 

The  plasmalogen  content  of  bull  semen  as  determined  by  Boguth 
(1952)  was  found  to  vary  from  30  to  90  mg./lOO  ml.;  of  this  about 
two-thirds  is  present  in  the  sperm  and  one-third  in  the  seminal 
plasma.  The  volume  taken  up  by  sperm  in  bull  semen  is  compara- 
tively small,  about  10%;  it  would  seem  therefore,  that  the  concentra- 
tion of  the  acetal  phospholipids  in  the  spermatozoa  themselves  must 
be  of  the  order  of  200-600  mg./lOO  g.  fresh  weight,  or  3  x  lO-^"  mg. 
per  cell. 


The  list  of  interesting  chemical  substances  which  occur  in  semen 
was  extended  again  when  in  1941  Wagner- Jauregg  reported  on  the 
isolation  of  the  hydrocarbon  heptacosane  from  human  semen.  An 
alcoholic  extract  obtained  from  18  litters  of  semen  formed,  upon 
concentration  in  vacuo,  a  solid  residue  which  was  extracted  first 
with  1-5  1.  acetone,  and  next  with  2  1.  of  a  mixture  of  equal  amounts 
of  ethanol  and  ether.  On  purification,  the  acetone-soluble  fraction 
yielded  some  crystalline  material  which  melted  at  57-60°  and  con- 
sisted, in  all  probability,  of  palmitic  and  stearic  acid.  The  ethanol- 
ether  soluble  fraction  formed  on  standing  a  crystalline  precipitate 
containing  1  g.  of  heptacosane,  CH3(CH2)25CH3,  which  on  recrystal- 
lization  showed  the  required  melting  point,  59-5°.  The  isolation  of 
heptacosane  has  previously  been  achieved  from  plant  material.  It  is 
also  known  to  be  associated  in  a  characteristic  manner  with  beeswax. 

128  The  Biochemistry  of  Semen 

So  far,  however,  the  only  instance  other  than  semen,  where  hepta- 
cosane  has  been  shown  to  occur  in  the  human  body,  is  in  the  urine 
of  pregnant  women.  It  is  absent  from  the  urine  of  men  and  of  non- 
pregnant women,  and  is  devoid  of  oestrogenic  activity.  Nothing 
is  known  about  the  origin  or  function  of  seminal  heptacosane. 
Should  future  investigations,  however,  show  that  heptacosane  in 
semen  is  involved  in  the  metabolism  of  fatty  acids  or  aldehydes,  then 
its  fate  would  be  analogous  to  that  of  plant  hydrocarbons  which 
are  well  known  to  be  associated  with  the  metabolism  of  fatty  acids, 
aldehydes  and  alcohols  in  plants. 

Role  of  lipids  in  sperm  jnetabolism 

The  functional  aspects  of  lipid  metabolism  in  spermatozoa  have 
been  the  subject  of  investigations  by  Lardy  and  Phillips  (1941«,  b\ 
1945).  To  begin  with,  these  authors  confirmed  the  observation 
originally  made  by  Redenz  (1933)  that,  in  contrast  to  whole  semen 
which  can  be  stored  successfully  both  anaerobically  and  aerobically 
owing  to  the  presence  of  glycolysable  carbohydrate  in  the  seminal 
plasma,  bull  spermatozoa  separated  from  the  seminal  plasma  by 
centrifugation  and  washing,  can  survive  only  in  the  presence  of 
oxygen.  From  this  they  inferred  that  when  the  spermatozoa  are 
deprived  of  sugar,  they  begin  to  oxidize  aerobically  some  of  their  own 
intracellular  constituents  as  a  source  of  energy  for  motility.  To 
detect  the  oxidizable  substrate,  sperm  samples  were  analysed  when 
fresh  and  after  periods  of  storage;  it  was  then  found  that  a  period 
of  aerobic  incubation  of  bull  spermatozoa  caused  a  significant  de- 
crease in  the  content  of  lipid  phosphorus  accompanied  by  an  increase 
in  the  acid-soluble  phosphorus.  But  when  glucose  was  added  to  the 
washed  sperm,  the  decrease  in  the  phospholipid  content  of  sperma- 
tozoa was  very  slight,  an  indication  perhaps,  of  a  preference  by 
spermatozoa  for  the  glycolytic  mechanism  as  a  source  of  energy 
(Table  18).  It  was  also  found  that  certain  phospholipids  prepared 
from  egg-yolk,  liver  and  soya  bean,  effectively  maintained  the 
oxygen  uptake  and  motility  of  washed  sperm  suspensions  under 
aerobic  conditions;  however,  on  the  addition  of  sugar  to  the  sperm 
suspension,  the  phospholipids  no  longer  produced  an  effect  on 
either  respiration  or  glycolysis. 

In  bull  semen  as  ejaculated,  with  its  large  reserve  of  readily 

Lipids  and  their  Role  in  the  Metabolism  of  Semen     129 

Table  18.  Changes  in  the  phospholipid  content  of  bull  spermatozoa 
in  presence  and  absence  of  sugar  (Lardy  &  Phillips,  \9A\a) 

(Bull  spermatozoa  freed  from  seminal  plasma  by  centrifugation,  then 
diluted  with  Ringer-phosphate  solution  to  the  original  volume  of  semen, 
and  incubated  at  room  temperature.) 

Medium  Phospholipid  content 

After  10  hr. 

Original  incubation 

(mg.  P/ml.)         (mg.  P/ml.) 

Ringer-phosphate  0-38  0-24 

Ringer-phosphate+0-04M-glucose  0-39  0-37 

glycolysable  material  in  the  form  of  fructose,  the  share  of  phos- 
pholipids in  sperm  metabolism  is  probably  small.  But  in  the  epi- 
didymis, where  glycolysable  sugar  is  unavailable,  Lardy  and  Phillips 
ascribe  great  importance  to  the  phospholipids  as  a  source  of  oxida- 
tive energy.  The  mechanism  of  utilization  of  this  reserve  is  held  to 
involve  hydrolytic  cleavage  of  phospholipids  foUov^ed  by  an  oxida- 
tion of  the  fatty  acid  portion  via  the  citric  acid  cycle,  and  coupled 
with  aerobic  phosphorylations.  According  to  Lardy,  Hansen  and 
Phillips  (1945),  the  aerobic  metabolism  of  phospholipids  in  the 
bovine  epididymal  spermatozoa  is  accompanied  by  an  uptake  of 
inorganic  phosphate,  and  the  formation  of  a  phosphate  ester  which 
is  hydrolysed  in  7  min.  by  N-HCl  at  100°,  and  thus  resembles 
adenosine  triphosphate. 

The  ability  to  utilize  phospholipids  as  a  source  of  aerobic  energy 
extends  to  the  spermatozoa  of  lower  animals,  notably  those  of  the 
sea-urchin.  One  of  the  main  differences  between  mammalian  and 
sea-urchin  semen  is  that  the  latter  contains  no  glycolysable  material 
in  the  seminal  plasma.  The  possibility  that  sea-urchin  spermatozoa 
which  have  been  shed  into  sea- water,  survive  at  the  expense  of 
energy  derived  from  the  oxidation  of  intracellular  carbohydrate  such 
as  glycogen,  also  appears  remote,  in  view  of  the  very  low  content 
of  glycogen-like  material  in  the  sperm  cells  (Stott,  1930;  Rothschild 
and  Mann,  1950).  On  the  other  hand,  according  to  Rothschild  and 
Cleland  (1952),  the  content  of  intracellular  phospholipids  which  in 
fresh  sperm  of  Echinus  esculentus  is  5-5%  of  the  dry  weight  or  4-14 
mg.  (0165  mg.  P)  per  10^*^  sperm  cells  decreases  in  the  course  of 
aerobic  incubation  of  sperm  suspensions  in  sea-water,  at  an  average 

130  The  Biochemistry  of  Semen 

rate  of  0-787  mg./lO^"  sperm  cells/7  hr.  The  oxygen  uptake  recorded 
during  the  same  period  is  1-45  ml.  Oa/lO^"  sperm,  which  if  sustained 
exclusively  by  phospholipids,  would  require  the  disappearance  of 
0-906  mg.  of  phospholipid.  On  the  basis  of  these  observations 
Rothschild  and  Cleland  conclude  that  the  principal  source  of  energy 
required  for  the  movement  of  sea-urchin  spermatozoa  is  derived 
from  the  oxidative  breakdown  of  phosphilipids  located  mainly  in 
the  middle-piece  of  the  sperm  cell. 

Lipids  in  the  seminal  plasma  and  male  accessory  gland  secretions 

Apart  from  the  lipids  which  form  a  part  of  the  sperm  structure, 
there  is  also  some  lipid  material  in  the  seminal  plasma.  The  bulk  of 
the  'bound  choline',  however,  does  not  consist  of  phospholipids 
but  occurs  in  the  form  of  acid-soluble  phosphorylated  derivatives  of 
choline  (see  p.  170).  The  lipid  of  the  human  seminal  plasma  origin- 

Table  19.  Lipids  of  the  human  prostatic  fluid  and  seminal  plasma 
(w^./lOO  ml.)  (Scott,  1945) 

(No.  indicates  the  number  of  studied  specimens.) 

Prostatic  fluid 

Seminal  plasma 



Lipid fraction             No. 








Total  lipid                    10 








Total  phosphatide       10 








Moist  ether-soluble 

phosphatides        10 








Lecithin                    10 








Cephalin                   10 








Moist  ether-insoluble 

phosphatides         10 








Total  cholesterol         10 








ates  chiefly  from  the  prostatic  fluid.  Moore,  Miller  and  McLellan 
(1941)  analysed  twelve  specimens  of  human  prostatic  secretion  and 
found  up  to  9-5  mg.  lipid  phosphorus  per  100  g.  fluid,  with  an 
average  of  2-7  mg.  P/100  g.  or  67-5  mg.  phospholipid/ 100  g.  Scott 
(1945),  whose  analytical  results  are  shown  in  Table  19,  found  an 
average  content  of  286  mg./lOO  ml.  of  'total  lipid'  and  179-8 
mg./lOO  ml.  of  phospholipid,  in  the  human  prostatic  secretion; 

Lipids  and  their  Role  in  the  Metabolism  of  Semen     131 

and  185-5  mg./lOO  ml.  of  'total  Upid'  and  83-5  mg./lOO  ml.  of 
phospholipid,  in  the  seminal  plasma.  However,  he  was  unable  to 
detect  lecithin  either  in  the  prostatic  fluid  or  in  the  seminal  plasma. 
In  both  instances,  two-thirds  of  the  phospholipid  consisted  of  an 
ether-soluble  choline-free  phosphatide,  probably  identical  with  cepha- 
line,  the  rest  being  some  other,  ether-insoluble  material.  Scott 
found  little  neutral  fat  in  either  the  prostatic  secretion  or  the  seminal 
plasma,  the  sum  of  phospholipids  and  cholesterol  accounting  for 
practically  the  entire  'total  lipid'.  The  content  of  70-120  mg.  total 
cholesterol /1 00  ml.  seminal  plasma  recorded  by  Scott,  is  below  the 
cholesterol  value  for  human  blood  plasma;  a  similar  figure,  80  mg./ 
100  ml.,  has  been  reported  earlier  by  Goldblatt  (1935a). 

'Lipid  bodies''  and  prostatic  calculi 

In  many  species,  the  seminal  plasma  contains  small  globules, 
droplets  or  granules,  sometimes  called  the  'lipid  bodies'.  In  man, 
dog,  cat,  and  rabbit,  they  are  derived  chiefly  from  the  prostatic 
secretion  but  in  certain  species  they  occur  also  in  the  seminal  vesicle 
secretion  (Prevost  and  Dumas,  1824;  Pittard,  1852).  The  globules  of 
the  human  prostatic  secretion  are  referred  to  by  Sir  Henry  Thompson 
in  his  famous  prize  essay  on  the  Diseases  of  the  Prostate  (1861), 
as  'small  yellowish  bodies,  in  appearance  sometimes  granular,  some- 
times homogeneous,  about  the  size  of  red  blood  corpuscles,  but  not 
so  uniform,  being  from  about  1/5000  to  1/2500  of  an  inch  in 
diameter'  and  exhibiting  'considerable  refractive  power  nearly  so 
much  as  to  give  them  a  resemblance  to  oil  globules'.  The  occurrence 
of  similar  elements  in  the  prostatic  secretion  was  later  observed  by 
Fuerbringer  (1881,  1886)  who  coined  for  them  the  name  Leclthin- 
kdrnchen\  These  'lecithin  granules'  or  'lecithin  bodies'  which  Fuer- 
bringer regarded  as  responsible  for  the  normal  opalescence  and 
milky  appearance  of  the  prostatic  fluid,  have  since  been  re-examined 
on  several  occasions,  mostly  by  means  of  histological  methods. 
Chemical  analysis  however,  failed  to  corroborate  the  presence  of 
lecithin  in  these  particles.  Other  curious  structures  which  according 
to  some  authors  are  closely  linked  with  the  appearance  of  'lipid 
bodies'  in  the  human  seminal  plasma,  are  certain  larger  bodies 
known  as  'colostrum  corpuscles',  'corpora  amylacea'  and  'pros- 
tatic calculi'.  The  colostrum  corpuscles,  frequently  met  with  in  the 

1 32  The  Biochemistry  of  Semen 

human  prostatic  secretion,  are  macrophages  packed  with  masses  of 
lipid  granules  which  stain  strongly  red  with  eosin.  The  corpora 
amylacea  are  small,  soft,  concentrically  laminated  spheroidal  bodies, 
pale  yellow  to  dark  brown  in  colour,  frequently,  though  not  invari- 
ably, doubly  refractile.  They  are  usually  located  in  the  larger  ducts 
and  acini  of  the  prostate  and  are  probably  made  up  of  desquamated 
epithelial  cells  and  prostatic  secretion.  They  have  been  shown  to 
contain  some  cholesterol  but  according  to  Moore  and  Hanzel 
(1936)  the  double  refraction  may  be  due  to  certain  purines,  decom- 
position products  of  nucleoproteins,  and  not  to  lipids.  The  prostatic 
calculi  are  ordinarily  not  more  than  a  few  mm.  in  diameter  but 
occasionally  they  may  replace  the  whole  prostatic  parenchyma. 
They  are  firm,  calcified  bodies,  the  basic  structure  of  which,  except 
for  size  and  infiltration  by  calcium  salts,  is  apparently  the  same  as 
that  of  the  corpora  amylacea  (Moore,  1936).  Wollaston  (1797) 
described  them  as  composed  of  'phosphorated  lime  in  the  state  of 
neutralization,  tinged  with  the  secretion  of  the  prostate  gland'.  He 
was  also  the  first  to  show  that  they  are  not  urinary  products.  In 
recent  times,  the  chemical  composition  of  prostatic  calculi  has  been 
investigated  by  Huggins  and  Bear  (1944);  a  considerable  proportion 
of  the  prostatic  stones  was  inorganic  and  consisted  of  calcium  and 
magnesium  phosphates  and  carbonates  but  there  was  also  some  21  % 
of  organic  matter  composed  of  protein,  citrate  and  cholesterol 
(Table  20). 

Although  characteristic  of  the  human  prostate,  corpora  amylacea 
are  also  found  elsewhere,  particularly  among  insectivores  where 
their  production  is  considered  to  be  one  of  the  chief  secretory 

Table  20.  Chemical  analysis  of  prostatic  calculi  (Huggins,  1947) 

(Stones  from  6  men;  average  values  expressed  in  %  of  dry  powdered 








Phosphorus  as  PO4 


Carbon  dioxide 


,                  Protein  (Nx  6-25) 


Citric  acid 




Lipids  and  their  Role  in  the  Metabolism  of  Semen     133 

functions  of  the  prostate  gland  (Hopkins,  1911;  Eadie,  1948^,  b). 
They  do  not  seem  to  occur  either  in  the  dog  or  the  rat.  The  total 
lipid  content  of  the  dog  prostatic  fluid  ranges  from  30  to  40 
mg./lOO  ml.  (Huggins,  1947)  and  the  lipid  phosphorus  from  M 
to  2-2  mg.  P/100  ml.  (Moore  et  al,  1941).  There  exists  a  condition 
known  as  the  'benign  prostatic  hypertrophy',  which  is  common  to 
dog  and  man.  In  the  dog,  however,  this  condition  is  not  associated 
with  the  occurrence  of  corpora  amylacea  or  any  other  spheroidal 
nodules  but  consists  of  cystic  hyperplasia  (Huggins  and  Clark, 
1940).  In  the  bull,  lipid-laden  cells  form  a  highly  characteristic 
component  of  the  seminal  vesicle  epithelium,  and  the  cavities  of 
the  tubules  in  the  seminal  vesicles  contain  an  abundance  of  eosino- 
philic granular  secretion  (Mann,  Davies  and  Humphrey,  1949).  The 
extent  to  which  organs  other  than  the  seminal  vesicle  and  prostate, 
contribute  to  the  lipid  or  sterol  content  of  semen,  has  not  been 
hitherto  studied  in  much  detail.  In  this  connection,  however,  an 
interesting  observation  of  Ward  and  Moore  (1953)  deserves  to  be 
mentioned,  concerning  the  occurrence  of  7-dehydrocholesterol  in 
the  preputial  gland  and  epididymis  of  rat. 


Fructose  and  Fructolysis 

Fructose  as  a  normal  constituent  of  semen.  Species  differences.  Site  of 
formation.  Seminal  fructose  as  an  indicator  of  male  sex  hormone  activity; 
the  'fructose  test'  and  its  application  to  certain  problems  of  sex  endocrin- 
ology. Role  of  hypophysis.  The  relationship  between  blood  glucose  and 
seminal  fructose.  Effect  of  malnutrition.  The  enzymic  mechanism  of  fruc- 
tose formation.  Anaerobic  and  aerobic  utilization  of  carbohydrate  by 
spermatozoa.  Pasteur  effect  and  the  'metabolic  regulator'.  Intermediary 
reactions  in  sperm  fructolysis  and  the  role  of  phosphorus-containing 

There  has  been  little  precise  knowledge  about  fructose  (laevulose) 
in  man  and  higher  animals  except  the  evidence  of  its  occurrence  in 
certain  embryonic  fluids  and  in  metabolic  dysfunctions  like  fruc- 
tosuria.  The  presence  of  a  laevorotatory  constitutent  in  foetal  fluids 
was  first  noted  by  Claude  Bernard  (1855)  but  its  chemical  identity 
was  not  recognized  until  some  time  later  when  it  was  shown  that 
fructose  was  a  normal  constituent  of  allantoic  and  amniotic  fluid, 
foetal  blood  and  the  urine  of  new-born  animals  (Majewski,  1858; 
Griiber  and  Griinbaum,  1904;  Paton,  Watson  and  Kerr,  1907; 
Langstein  and  Neuberg,  1907;  Orr,  1924;  Cole  and  Hitchcock, 
1946;  Bacon  and  Bell,  1948;  Hitchcock,  1949).  More  recently,  the 
source  of  foetal  fructose  was  traced  to  the  placenta  (Huggett, 
Warren  and  Warren,  1951).  So  far  as  adult  man  is  concerned,  it 
was  believed  that  in  general  the  occurrence  of  fructose  is  restricted 
to  pathological  conditions;  fructose  has  been  demonstrated  in 
transsudates,  and  in  the  urine  of  diabetics  and  persons  suffering 
from  the  peculiar  metabolic  disorder  known  as  'spontaneous  fruc- 
tosuria',  the  aetiology  of  which  remains  obscure.  In  the  normal 
human  or  animal  organism,  fructose  has  been  found  to  be  utilized 
chiefly  after  enzymic  conversion  to  glucose  and  glycogen;  liver, 
kidney  and  the  gastro-intestinal  tract  were  shown  to  be  the  main 
sites  of  this  process  (Oppel,  1930;  Bollman  and  Mann,  1931; 
Stewart   and   Thompson,    1941;   Deuel,    1936;    Reinecke,    1944). 


Fructose  and  Fructolysis  135 

Thus,  in  the  light  of  the  evidence  available  until  relatively  recently, 
it  seemed  rather  improbable  that  in  the  normal,  fully-developed 
mammalian  organism,  fructose  could  occupy  a  place  on  the  list 
of  'animal  carbohydrates',  or  that  any  specific  function  could  be 
assigned  to  this  sugar. 

Fructose  as  a  normal  constituent  of  semen 

Since  the  early  researches  on  mammalian  semen  by  McCarthy, 
Stepita,  Johnston  and  Killian  (1928),  Ivanov  (1931),  Huggins  and 
Johnson  (1933)  and  other  pioneers  in  the  field  of  semen  biochemistry, 
it  was  known  that  in  several  species,  including  man,  a  reducing  and 
yeast-fermentable  sugar  is  normally  present  in  semen,  the  concen- 
tration of  this  sugar  exceeding  by  far  that  of  glucose  in  blood. 
However,  up  to  1945,  in  the  extensive  literature  dealing  with  the 
subject  of  seminal  sugar,  this  substance  has  been  described  either  as 
glucose  or  simply  as  the  reducing  sugar  of  semen  (Killian,  1933; 
Bernstein,  1933;  Goldblatt,  1935«;  Shergin,  1937;  McKenzie,  Miller 
and  Bauguess,  1938;  Davis  and  Cole,  1939;  Moore  and  Mayer, 
1941;  MacLeod  and  Hotchkiss,  1942;  Salisbury  and  VanDemark, 
1945),  and  the  only  reference  to  a  probable  occurrence  of  fructose 
in  semen  is  found  in  an  early  paper  by  Yamada  (1933)  who  in  a 
general  survey  of  human  tissues  and  body  fluids  carried  out  numer- 
ous fructose  determinations  by  means  of  a  colour  reaction  with  the 
drug  'cryogenine';  of  course,  like  so  many  colour  tests,  this  reaction 
by  itself  cannot  be  regarded  as  specific  for  fructose,  since  it  gives 
a  positive  result  not  only  with  fructose  but  also  with  other  ketoses, 
nor  does  it  distinguish  between  free  fructose,  that  is  D(-)-fructo- 
pyranose  (formula  in  Fig.  14),  and  bound  fructose,  i.e.  fructofura- 
nose,  such  as  occurs  for  example,  in  the  various  phosphofructoses . 

In  1945,  in  the  course  of  studies  on  the  metabolism  of  semen,  the 
seminal  sugar  was  isolated  for  the  first  time  and  identified  by  chemi- 
cal methods  as  free  D(-)-fructose  (Mann,  \9A5b\  \946a,  b,  c).  The 
actual  final  isolation  was  accomplished  with  a  120  ml.  sample  of  bull 
semen  representing  some  thirty  pooled  bull  ejaculates,  which  were 
collected  within  a  twelve-hour  period  by  the  various  Centres  for 
Artificial  Insemination  of  Cattle  in  England,  and  immediately  des- 
patched to  Cambridge.  The  chemical  procedure  involved  the  fol- 
lowing steps:  (a)  the  preparation  of  methylphenyl-fructosazone,  a 

136  The  Biochemistry  of  Semen 

crystalline  compound  which  has  been  shown  by  Neuberg  (Neuberg, 
1902,  1904;  Neuberg  and  Strauss,  1902;  Langstein  and  Neuberg, 
1907;  Neuberg  and  Mandl,  1946)  to  be  one  of  the  few  chemical 
derivatives  by  means  of  which  fructose  can  be  identified  and  dis- 
tinguished from  glucose  and  from  other  closely  related  sugars;  {b)  the 
purification  of  seminal  fructose  up  to  the  point  when  it  reached  the 
specific  optical  activity  of  pure  crystalline  fructose:  [aP°°=  -92-2°; 
(c)  the  demonstration  that  fructose  occurs  in  the  semen  in  free  form 
and  that  it  accounts  for  the  whole  of  the  yeast-fermentable  carbo- 
hydrate which  yields  'ketose  reactions'  with  resorcinol  (Seliwanoff, 
1887;  Roe,  1934),  diphenylamine  (Ihl,  1885)  and  similar  colour- 
producing  substances  (Pinoff,  1905;  Thomas  and  Maftei,  1927; 
Pryde,  1946);  {d)  proof  obtained  with  the  highly  specific  enzyme, 
glucose  oxidase,  that  in  semen  glucose  is  either  absent  or  present  in 
mere  traces. 

On  the  basis  of  the  above  findings,  which  excluded  the  presence 
of  glucose,  bound  fructose,  and  other  ketoses,  a  rapid  colorimetric 
method  has  been  developed  by  means  of  which  it  is  possible  to 
determine  accurately  the  fructose  content  of  semen;  0  05-0  1  ml. 
suffices  for  analysis  of  human,  bull,  ram  or  rabbit  semen  (Mann, 
1948a,  b\  1952). 

Species  differences 

The  following  mammalian  species  have  been  found  to  contain 
fructose  in  semen:  man,  bull,  ram,  boar,  stallion,  goat,  opossum, 
rabbit,  guinea-pig,  rat,  mouse,  hamster  (Mann,  1949).  Among  the 
lower  animals,  fructose  was  found  in  the  semen  of  an  elasmobranch 
(the  dogfish,  Scylliorhinus  caniculus)  and  in  the  reproductive  organs 
of  the  male  (but  not  female)  grasshopper,  Locusta  migratoria 
(Humphrey  and  Mann,  1948;  Humphrey,  1949).  In  this  connection 
it  is  worthwhile  to  recall  the  occurrence  of  fructose  in  the  haemo- 
lymph  of  the  larvae  of  another  insect,  Gastrophiliis  intestinalis 
(Levenbook,  1947). 

There  are,  however,  considerable  quantitative  differences  between 
the  various  species.  In  the  bull  and  goat,  for  example,  the  con- 
centration of  fructose  in  semen  sometimes  reaches  a  level  of 
1000  mg./lOO  ml.,  but  in  the  boar  and  stallion  it  seldom  exceeds 
50  mg./lOO  ml.  Human  semen  occupies  an  intermediate  position  as 

Fructose  and  Fructolysis  137 

can  be  seen  from  Tables  4  and  5,  which  include  values  for  fructose 
in  several  species.  But,  when  comparisons  are  made  between  a  species 
with  fructose-rich  semen  (bull)  and  one  notoriously  poor  in  seminal 
fructose  (boar),  it  must  not  be  forgotten  that  the  volume  of  a  single 
boar  ejaculate  is  almost  a  hundred  times  that  of  a  bull,  so  that  in 
effect,  a  single  ejaculate  of  either  species  contains  about  the  same 
absolute  amount  of  fructose.  There  are  species,  however,  in  which 
fructose  is  altogether  absent  from  semen  or  present  only  in  traces, 
and  it  is  through  the  study  of  these  animals  that  we  may  hope  to 
gain  insight  into  the  problem  of  alternative  sugars  in  semen.  Cock 
semen  for  example,  has  no  fructose  or  a  negligible  amount  only,  but 
it  contains  a  certain  amount  (20-100  mg./lOO  ml.)  of  anthrone- 
reactive  material  of  which  a  variable  fraction  disappears  on 
oxidation  with  glucose  oxidase  and  must  therefore,  be  identical 
with  glucose  (Mann  and  Hancock,  1952).  Rabbit  semen,  unlike 
that  of  bull,  ram  and  man,  contains  occasionally  an  appreciable 
admixture  of  glucose  in  addition  to  fructose  (Mann  and  Parsons, 

Site  of  formation 

The  reason  for  the  conspicuous  species  differences  in  the  concen- 
tration of  fructose  as  well  as  the  individual  fluctuations  (Table  4 
and  5),  is  the  fact  that  fructose  is  a  product  not  of  the  testes,  but 
of  the  male  accessory  organs  of  reproduction,  principally  the  seminal 
vesicles  (Mann,  \9A6b).  Naturally,  the  highly  variable  anatomical 
characteristics  of  these  glands  such  as  their  size,  actual  storage 
capacity,  and  secretory  ability,  are  important  factors  which  deter- 
mine the  final  output  of  fructose  in  the  ejaculate  (Fig.  4).  All  these 
considerations  are  pertinent  to  studies  of  human  semen  because  of 
the  exceptionally  large  individual  variations  in  the  secretory  func- 
tion of  the  seminal  vesicles  and  their  rather  small  storage  capacity 
which  explains  why  the  collection  of  consecutive  ejaculates  within 
a  few  days,  usually  yields  samples  with  a  conspicuously  low  level 
of  fructose.  It  appears  that  a  time  interval  of  about  two  days  is 
required  to  replenish  the  store  of  fructose  in  the  vesicular  secretion  of 
man.  Unlike  in  certain  other  mammals,  the  human  seminal  vesicle 
and  vas  deferens  open  into  the  urethra  through  a  common  channel 
known  as  the  ejaculatory  duct.  Consequently,  any  obstruction  at  the 

138  The  Biochemistry  of  Semen 

level  of  the  ejaculatory  ducts  will  prevent  both  fructose  and  sperma- 
tozoa from  reaching  the  urethral  canal.  This  fact  has  been  aptly 
chosen  as  an  aid  to  medical  diagnosis  by  Young  (1948,  1949) 
who  described  the  case  of  a  man  in  whom  repeated  semen  analysis 
failed  to  detect  fructose  or  sperm,  although  testicular  biopsy  re- 
vealed normal  spermatogenesis;  the  case  has  been  diagnosed  as 
congenital  bilateral  aplasia  of  the  vasa  deferentia. 

It  must  be  also  mentioned  that  though  the  seminal  vesicles  are 
the  main  source  of  fructose  in  the  higher  mammals,  yet  an  addi- 
tional small  amount  of  this  seminal  sugar  is  derived  from  the 
ampullar  glands  (Mann,  1948^),  and  in  some  animals  also  from 
certain  other  glands.  Thus,  in  the  rabbit,  fructose  was  located  both 
in  the  glandula  vesicularis  (a  structure  corresponding  to  seminal 
vesicles)  and  in  the  ampullae,  as  well  as  in  the  prostate  (Davies  and 
Mann,  \9Alb).  The  rat  provides  an  instance  of  particular  interest, 
as  in  this  rodent  the  seminal  vesicles  are  free  from  fructose  alto- 
gether; instead,  fructose  is  found  in  the  dorso-lateral  prostate  and 
in  the  so-called  coagulating  gland,  a  small  organ  immediately 
adjacent  to  the  seminal  vesicles  proper,  with  which  it  shares  a 
common  peritoneal  sheath  (Humphrey  and  Mann,  1948,  1949). 

Since  fructose  is  produced  by  the  accessory  glands,  and  not  the 
testes,  it  is  not  surprising  that  in  whole  fresh  semen  there  is  no 
direct  proportion  between  fructose  concentration  and  sperm  density. 
On  the  contrary,  both  in  man  and  in  domestic  animals,  an  inverse 
ratio  between  fructose  and  sperm  concentration  in  semen  is  fre- 
quently met  with;  the  simplest  interpretation  is  that  in  a  dense 
sample  of  semen  the  space  occupied  by  the  sperm  cells  is  relatively 
larger,  and  the  volume  taken  up  by  the  fluid  portion,  i.e.  the 
fructose-containing  seminal  plasma,  correspondingly  less.  This 
factor  has  a  direct  bearing  on  the  interpretation  of  laboratory 
examinations  concerned  with  semen  and  male  fertility  or  sterility. 
It  explains,  for  instance,  why  a  semen  sample  with  a  high  content 
of  fructose  need  not  necessarily  be  one  of  good  sperm  quality,  and 
furthermore,  why  it  is  possible  to  come  across  samples  with  a  high 
fructose  content  but  of  low  sperm  density.  In  fact,  some  of  our 
highest  values  for  fructose  so  far  recorded,  were  encountered  in 
the  semen  of  vasectomized,  and  thus  completely  azoospermic, 

Fructose  and  Fructolysis  139 

Seminal  fructose  as  an  indicator  of  male  sex  hormone  activity;  the 
'fructose  test^  and  its  application  to  certain  problems  of  sex 

The  'fructose  test',  originally  described  by  Mann  and  Parsons 
(1947)  and  subsequently  developed  by  Mann,  Davies  and  Hum- 
phrey (1949),  Mann,  Lutwak-Mann  and  Price  (1948)  and  Mann 
and  Parsons  (1950),  is  founded  on  the  observation  that  the  capacity 
of  the  accessory  organs  to  produce  fructose  and,  thereby,  the  actual 
level  of  fructose  in  the  seminal  plasma,  reflects  in  a  faithful  manner 
the  degree  of  testicular  hormone  activity  in  the  male,  and  in  this 
way  provides  an  accurate  indicator  of  endocrine  testicular  function. 
In  experiments  on  rats  and  rabbits  it  was  shown  that  seminal  fruc- 
tose disappears  almost  completely  within  two  weeks  after  castration 
and  also  that  the  postcastrate  fall  in  the  level  of  fructose  can  be 
prevented,  or,  if  already  developed,  fully  restored,  by  the  implan- 
tation of  testosterone  (Fig.  12).  The  effect  is  not  limited  to  labora- 
tory animals  and  analogous  results  were  obtained  with  domestic 
animals  such  as  the  bull. 

The  test  can  be  carried  out  in  two  ways,  by  the  chemical  analy- 
sis of  the  seminal  fluid  collected  from  an  intact  animal  by  means 
of  an  artificial  vagina,  or  by  the  analysis  of  accessory  organs  of 
reproduction  obtained  from  the  experimental  animal  by  dissection. 
The  first  method  gives  an  opportunity  to  observe  in  the  same  animal 
the  time-sequence  of  changes  brought  about  by  castration  and  hor- 
monal treatment,  and  eliminates  the  sacrifice  of  the  experimental 
animal.  In  the  second  procedure,  on  the  other  hand,  the  test  can 
be  used  for  a  quantitative  assay  of  male  sex  hormone  activity  in  the 
whole  body,  isolated  tissues,  body  fluids  and  hormone  preparations; 
as  an  illustration.  Fig.  13  gives  a  dosage-response  curve  obtained 
with  coagulating  glands  of  castrated  rats  which  were  injected  for 
three  weeks  with  known  doses  of  testosterone  propionate;  following 
the  last  day  of  injections  the  rats  were  sacrificed,  the  coagulating 
glands  dissected  and  used  for  fructose  analysis. 

Below  are  discussed  some  of  the  endocrinological  problems  to 
which  an  approach  was  made  in  recent  years  with  the  aid  of  the 
'fructose  test',  applied  either  alone  or  in  conjunction  with  the  'citric 
acid  test',  which  depends  on  the  relationship  between  the  secretion 


The  Biochemistry  of  Semen 

of  citric  acid  by  some  of  the  accesory  organs  and  the  male  sex 
hormone  activity. 


Pellet  implanted 

Pellet  removed 

2       3       4        5       6       7        8 
Weeks  after  castration 
Fig.  12.  Post-castrate  fall  and  testosterone-induced  rise  of  seminal  fructose 
in  rabbit;  pellet:  100  mg.  testosterone. 

(Mann  &  Parsons,  1947) 

{a)  Time  relationship  between  the  onset  of  secretory  activity  in  male 
accessory  glands  and  spermatogenesis.  In  young  rabbits  (Davies  and 
Mann,  1941  b),  rats  (Mann,  Lutwak-Mann  and  Price,  1948),  bull- 
calves  (Mann,  Davies  and  Humphrey,  1949)  and  boars  (Mann,  1954), 

Fructose  and  Fructolysis 


fructose  and  citric  acid  appear  in  the  accessory  glands  at  an  early 
age,  before  there  is  any  evidence  of  active  spermatogenesis;  since 
the  secretion  of  both  these  substances  depends  upon  the  presence  of 
the  male  sex  hormone,  it  must  be  concluded  that  the  hormone 
begins  to  function  in  the  male  body  well  in  advance  of  the  actual 
spermatogenesis.   Thus,   for  instance,   in  bull-calves  appreciable 

0     5  25  50 

Daily  dose  of  testosterone  propionate  (/ig.) 

Fig.  13.  Dosage-response  curves  of  testosterone  propionate,  using  the  coagu- 
lating glands  of  the  rat;  0 -O,  fructose  (/^tg.);  • •, 

weight  of  organs  (mg.). 

(Mann  &  Parsons,  1950) 

amounts  of  fructose  are  found  in  the  vesicular  secretion  already  at 
the  age  of  about  four  months,  whereas  the  first  mature  spermatozoa 
appear  nearly  eight  months  later.  One  cannot,  of  course,  rule  out 
the  possibility  that  the  testicular  hormone  is  active  in  the  bull-calf 
even  before  the  age  of  four  months,  but  if  so,  then  either  its  con- 
centration is  too  small  to  produce  a  distinct  response  in  the  accessory 
organs,  or  else  its  action  is  countered  by  some  other  factors. 
ib)  Effect  of  testosterone  on  the  appearance  of  fructose  in  castrated 

142  The  Biochemistry  of  Semen 

animals.  The  following  experiment  was  carried  out  by  Mann,  Davies 
and  Humphrey  (1949)  at  the  Agricultural  Research  Council  Field 
Station  at  Compton,  in  Berkshire.  Six  bull-calves  were  used.  These 
were  castrated  when  one  to  two  weeks  old,  i.e.  at  an  age  prior  to  the 
appearance  of  fructose  in  the  seminal  glands.  Seven  months  later 
two  of  the  castrated  calves  received  subcutaneous  implants  of  0-5  g. 
pellets  of  pure  testosterone,  whereas  the  remaining  four  were  left 
untreated.  After  another  four  weeks  all  six  animals  were  sacrificed 
and  their  seminal  glands  dissected  out,  weighed,  and  examined  both 
chemically  and  histologically.  The  unused  portions  of  the  hormone 
pellets  were  recovered  from  the  subcutaneous  tissue  of  the  two 
hormone-treated  calves;  their  weights  were  0-344  and  0-338  g. 
respectively,  showing  that  the  quantities  of  testosterone  absorbed 
per  month  per  animal  were  0- 1 56  and  0- 1 62  g.,  respectively.  Chemical 
analysis  revealed  the  presence  of  considerable  amounts  of  fructose 
in  the  seminal  glands  in  response  to  the  four  weeks'  hormone  treat- 
ment (51  mg.  fructose  per  100  g.  tissue  or  5-3  mg.  fructose  per 
total  gland),  as  against  a  negligible  fructose  content  in  the  untreated 
castrates  (8  mg.  per  100  g.  or  0-25  mg.  per  total  gland).  In  compari- 
son with  and  in  contrast  to  the  marked  chemical  difference,  the 
evidence  for  the  functional  recovery  in  the  seminal  glands,  as 
assessed  by  the  histological  examination,  was  practically  impercep- 
tible (Plate  VI).  In  this  way  we  were  able  to  provide  evidence  that 
the  early  effects  of  testosterone  treatment  can  be  established  far 
more  convincingly  by  the  large  percentage-increase  in  the  fructose 
content  of  the  seminal  gland  secretion,  than  by  means  of  histological 
methods  which  at  this  stage  failed  to  show  significant  changes  in  the 
glandular  tissue. 

An  investigation  concerned  with  the  response  to  testosterone  was 
also  made  by  Rudolph  and  Samuels  (1949)  on  rats,  and  by  Gassner 
and  his  co-workers  (1952)  on  bulls.  In  castrated  rats,  a  significant 
increase  in  the  fructose  content  of  accessory  organs  was  noticed 
already  ten  hours  after  the  injection  of  1  mg.  testosterone  pro- 
pionate. In  bulls,  fructose  disappeared  from  ejaculates  within  two 
weeks  after  castration  but  injections  of  testosterone  propionate,  if 
given  within  four  weeks  after  castration,  led  to  a  rapid  return  of 
fructose  production  to  the  pre-castrate  level;  yet,  in  spite  of  the  fully 
restored  fructose  level,  such  seminal  plasma,  when  added  to  washed 













Histological  sections  from  a  tubule  (mag. x 437),  and  the  fructose  content 
of  seminal  vesicle. 

A.  from  a  bull-calf  castrated  when  three  weeks  old,  and  killed  when  nine 

months  old. 

B.  from  a  bull-calf  castrated  when  three  weeks  old,  left  untreated  till 

eight  months  old,  and  then  implanted  with  testosterone  (0-5  g.); 
killed  one  month  later,  simultaneously  with  calf  A. 

Fructose  and  Fructolysis  143 

spermatozoa  obtained  from  a  normal  bull,  was  unable  to  support 
sperm  metabolism  to  the  same  extent  as  plasma  from  normal  i.e. 
non-castrated  animals. 

An  interesting  example  of  the  application  of  the  fructose  test  to 
problems  of  infertility  in  man  has  been  provided  by  a  study  of  four 
eunuchoid  patients  who  responded  to  androgenic  treatment  with  a 
highly  significant  elevation  of  fructose  in  semen  (Landau  and 
Longhead,  1951). 

It  seems  probable  that  the  fluctuations  of  fructose  level  in  the 
semen  of  normal  individuals  may  also  be  due,  in  part  at  least,  to 
some  periodic  changes  in  the  activity  of  the  testicular  hormone  in 
the  male  body.  Normal  rats,  injected  with  large  doses  of  the  male 
hormone  invariably  react  by  an  increased  level  of  fructose  formation, 
well  above  that  of  non-treated  controls.  The  effect  is  particularly 
striking  with  breeds  of  animals  which  exhibit  a  relatively  low  physio- 
logical level  of  fructose  formation.  In  this  connection,  however,  it  is 
interesting  to  note  that  when  injections  of  large  doses  of  testosterone 
propionate  into  normal  rats  are  continued  to  excess,  e.g.  200  i-ig. 
daily  for  forty  days,  the  state  of  overstimulation  in  the  accessory 
organs  is  accompanied  by  a  marked  decline  in  the  size  of  the  testes; 
after  seven  weeks  of  such  treatment  the  reduction  in  the  weight  of 
the  testes  is  nearly  50%  (Mann  and  Parsons,  1950).  Injections  of 
excessive  doses  of  androgens  are  well  known  to  produce  harmful 
effects  on  the  spermatogenesis  in  animals  and  in  man  (Moore,  1939; 
McCullagh  and  McGurl,  1939;  Meckel,  1951). 

In  normal  bulls,  a  dose  of  100  mg.  testosterone  propionate, 
repeated  three  times  weekly  for  six  weeks,  appears  to  produce  only 
a  very  slight  increase  in  the  level  of  fructose  in  semen  (Gassner, 
Hill  and  Sulzberger,  1952).  However,  according  to  another  report, 
sexual  excitation  prior  to  service  has  a  stimulating  effect  on  the  out- 
put of  fructose  in  bull  semen  (Branton,  D'Arensbourg  and  Johnston, 

(c)  Hormone-induced  formation  of  fructose  in  subcutaneous  trans- 
plants from  accessory  organs.  Once  the  dependence  of  seminal  fruc- 
tose upon  the  activity  of  the  male  sex  hormone  had  been  established 
it  was  possible  to  enquire  into  the  mechanism  of  this  hormonal  rela- 
tionship. One  of  the  problems  to  settle  was  the  extent  to  which  the 
process  of  fructose  generation  in  accessory  glands  depends  upon  the 

144  The  Biochemistry  of  Semen 

preservation  of  intact  vascular  and  neural  links.  Insight  into  this 
matter  was  gained  by  the  technique  of  subcutaneous  transplantation, 
when  it  was  demonstrated  that  small  fragments  of  rat  coagulating 
gland,  about  1  mg.  in  weight,  transplanted  subcutaneously  into 
normal  adult  male  hosts,  grew  well  and  showed  after  some  weeks 
of  subcutaneous  development  a  high  content  of  fructose.  Follow- 
ing castration  of  the  hosts,  the  transplants  lost  their  ability  to 
form  fructose  but  this  was  promptly  restored  by  treatment  with 
testosterone  propionate.  Perhaps  the  most  remarkable  fact  in  these 
experiments  was  that  the  growth  of  the  grafts  and  their  chemical 
secretory  function  occurred  not  only  in  male,  but  also  in  female 
hosts  provided  that  the  latter  were  treated  with  testosterone 
(Lutwak-Mann,  Mann  and  Price,  1949). 

Thus,  for  the  first  time  the  effect  of  the  male  sex  hormone  on 
fructose  secretion  was  demonstrated  in  tissue  fragments  dissected 
from  the  male  accessory  organs  and  developing  in  complete  isola- 
tion from  the  rest  of  the  male  generative  system.  Actually,  the  trans- 
plants had  an  even  higher  fructose  content  than  the  corresponding 
intact  glands  of  the  graft-bearing  hosts,  because  unlike  intact  glands, 
the  grafts  lack  a  secretory  outlet. 

In  another  study,  Price,  Mann  and  Lutwak-Mann  (1949,  1954) 
applied  the  transplantation  technique,  coupled  with  the  chemical 
methods,  to  the  problem  of  the  androgenic  activity  of  ovarian 
hormones  in  the  female  rat.  Subcutaneous  transplants  of  rat  coagu- 
lating gland  in  female  hosts  were  shown  to  secrete  large  quantities 
of  fructose  in  response  to  injections  of  pregnant  mare  serum  gona- 
dotrophin.  A  series  of  thirty  injections  of  twenty  international  units 
of  equine  gonadotrophin  was  given  daily;  at  autopsy  the  ovaries  of 
the  female  hosts  were  enlarged  at  least  tenfold  and  covered  with 
numerous  follicles  and  corpora  lutea.  In  these  rats,  gonadotrophin, 
through  a  stimulating  action  on  the  ovaries,  raised  the  output  of 
ovarian  androgens  to  an  extent  which  induced  the  secretion  of  fruc- 
tose in  transplants  from  the  coagulating  gland. 

{d)  Effects  of  progesterone,  stilboestrol  and  oestradiol.  The  nature 
of  the  ovarian  androgen  responsible  for  the  formation  of  fructose 
is  unknown,  but  there  are  indications  that  it  may  be  related  to 
progesterone  or  to  a  product  of  progesterone  metabolism.  An 
inquiry  into  the  androgenic  value  of  progesterone  showed  that  large 

Fructose  and  Fructolysis  145 

doses  of  progesterone  injected  into  castrated  male  rats  have  a 
definite  androgenic  effect;  it  was  calculated  that  the  androgenic 
value  of  25  mg.  pure  progesterone  is  slightly  more  than  that  of 
0005  mg.  testosterone  propionate  (Price,  Mann  and  Lutwak-Mann, 
1949,  1954). 

Whereas  progesterone  exhibits  some  androgenic  activity,  stil- 
boestrol  is  endowed  with  the  properties  of  an  androgen-antagonist. 
The  testosterone-catalysed  secretion  of  fructose  in  the  male  accessory 
gland  secretions  of  a  rabbit  can  be  suppressed  very  effectively  by  the 
subcutaneous  implantation  of  25  mg.  stilboestrol  (Parsons,  1950). 
In  experiments  in  which  a  castrated  rabbit  received  simultaneously 
implants  of  testosterone  and  stilboestrol,  the  latter  prevented  com- 
pletely the  production  of  fructose  by  the  accessory  organs. 

It  appears  that  in  castrated  bulls,  small  amounts  of  oestradiol 
dipropionate  used  together  with  testosterone,  have  a  small  but 
definite  synergistic  effect  on  the  seminal  vesicles  and  lead  to  a  higher 
output  of  fructose  in  semen  (Gassner,  Hill  and  Sulzberger,  1952). 

Role  of  hypophysis 

The  endocrine  influence  of  the  testes  on  the  formation  of  fructose 
in  accessory  organs  is  integrated  closely  with  the  functioning  of  the 
anterior  pituitary  gland.  Hypophysectomy,  like  gonadectomy,  invari- 
ably results  in  a  rapid  decline  in  the  level  of  fructose  in  the  seminal 
plasma  (Mann  and  Parsons,  1950).  In  the  rabbit,  for  instance,  a 
three  to  four  weeks'  period  after  castration  or  hypophysectomy  alike, 
usually  leads  to  complete  disappearance  of  fructose  so  that  an 
ejaculate  collected  by  means  of  an  artificial  vagina  three  weeks 

Table  21.  Ejfect  of  testosterone  and  pregnant  mare  serum 
gonadotrophin  on  the  formation  of  fructose  in  rabbit  prostate 

Weight  of  Fructose 

prostate  content 

Rabbit  (mg.)  (//g. /organ) 

1.  Normal  770  935 

2.  3  weeks  after  castration  280  20 

3.  6  weeks  after  castration  and  simultaneous 

implantation  of  testosterone  (100  mg.)  1000  1220 

4.  4  weeks  after  hypophysectomy  149  10 

5.  6  weeks  after  hypophysectomy;  for  the  last 

4  weeks  injected  200  I.U.  PMS-gonado- 

trophin  210  395 

146  The  Biochemistry  of  Semen 

after  the  operation  contains  no  more  than  20  ^g.  fructose,  as  com- 
pared with  500  to  1000  /^g.,  before  the  operation.  Both  castrated  as 
well  as  hypophysectomized  animals  promptly  respond  to  the  sub- 
cutaneous implantation  or  injection  of  testosterone  with  renewed 
secretion  of  fructose.  The  same  happens  if  instead  of  testosterone 
pregnant  mare  serum  gonadotrophin  is  injected  into  a  hypophy- 
sectomized animal  (Table  21). 

The  relationship  between  blood  glucose  and  seminal  fructose 

In  addition  to  the  hormones  of  the  testis  and  the  pituitary  gland, 
yet  another  organ,  the  pancreas,  exerts  a  profound  influence  upon 
the  level  of  fructose  in  semen.  The  effect  is  an  indirect  one,  and  is 
brought  about  by  the  action  of  insulin  on  the  level  of  blood  glucose 
which  in  turn  governs  the  level  of  fructose  in  semen.  The  existence 
of  a  causal  link  between  the  blood  sugar  level  and  seminal  fructose 
was  studied  at  first  in  animals  with  experimental  diabetes;  later, 
however,  it  was  also  shown  in  diabetic  man  (Mann  and  Parsons, 
1949,  1950). 

In  rabbits,  experimental  diabetes  can  be  produced  with  alloxan; 
best  results  are  obtained  by  injecting  intravenously  into  a  rabbit 
75  mg.  alloxan  per  kg.  body  weight,  and  repeating  this  dose  one  or 
two  days  later.  Fig.  14  illustrates  the  course  of  such  an  experiment 
with  a  rabbit  in  which  analyses  of  blood  and  semen  were  carried 
out  regularly  during  a  period  of  four  months.  At  the  outset  of  the 
experiment  this  animal  had  a  blood  glucose  content  of  100  mg.  per 
100  ml.,  and  about  600  i-ig.  fructose  per  ejaculate  or  70  mg.  per 
100  ml.  semen  (fluid  portion).  However,  following  alloxan  treatment, 
the  rabbit  developed  severe  glucosuria  within  two  days  and  its  blood 
glucose  level  rose  to  350  mg./lOO  ml.;  at  the  same  time,  the  level  of 
fructose  in  semen  began  to  increase,  until  three  weeks  later  there 
was  500  mg.  glucose  per  100  ml.  blood,  and  3500  /^g.  fructose  per 
ejaculate  or  320  mg.  fructose  per  100  ml.  semen.  When  it  was  estab- 
lished that  hyperglycaemia  is  followed  by  an  increased  concentra- 
tion of  fructose  in  semen,  the  effect  of  insulin  was  examined  and  it 
was  found  that  the  insuUn-induced  fall  in  blood  glucose  was  followed 
by  a  reduction  in  the  fructose  content  of  semen;  moreover,  once  the 
effect  of  insulin  on  blood  glucose  in  the  diabetic  animal  wore  off, 
there  was  again  an  increase  in  seminal  fructose. 

Fructose  and  Fructolysis  147 

Conditions  similar  to  those  in  experimental  diabetes  seem  to 
prevail  also  in  man.  In  semen  samples  from  diabetic  patients  we 

8         lO       12 


Fig.  14.  Effect  of  diabetes  and  insulin  on  seminal  fructose  in  rabbit.  The 
period  of  insulin  treatment  is  indicated  by  arrows.  Semen  was  col- 
lected weekly. 

came  upon  fructose  values  which  were  well  above  the  upper  limit 
of  normal  variations;  diabetic  values  ranged  from  650  to  1230  mg. 
per  100  ml.,  and  from  33-4  to  47-5  mg.  per  ejaculate.  In  a  survey 
of  150  specimens  of  normal  human  semen,  Harvey  (1948)  found 

148  The  Biochemistry  of  Semen 

640  mg.  per  100  ml.  or  31-6  mg.  per  ejaculate  to  be  the  highest 
value.  It  is  interesting  to  recall  here  that  years  ago  Goldblatt  (1935a) 
noticed  a  high  reducing  sugar  value  in  human  diabetic  semen  but 
attributed  this  to  urinary  glucose. 

Effect  of  malnutrition 

It  has  long  been  known  that  defective  nutrition  has  a  deleterious 
influence  upon  the  male  reproductive  system.  One  of  the  earliest 
surveys  of  this  problem  is  found  in  the  monograph  by  Jackson  (1 925); 
this  was  followed  by  the  work  on  degenerative  changes  in  testes  and 
sterility  associated  with  vitamin  A  and  E  deficiency  and  in  later 
years,  by  many  other  nutritional  studies  which  helped  to  accumulate 
a  wealth  of  information  on  this  subject,  fully  reviewed  on  several 
occasions  (Asdell,  1949;  Burrows,  1949;  Lutwak-Mann,  1951; 
Mason,  1949;  Reid,  1949;  Russell,  1948;  Samuels,  1948;  Walton, 
1949).  Most  investigators  in  this  field,  however,  particularly  those 
concerned  with  problems  of  human  fertility,  were  much  more  in- 
terested in  the  spermatogenic  activity  of  the  testicular  tissue  than 
in  the  function  of  the  accessory  organs  of  reproduction.  It  was, 
therefore,  something  of  a  departure  when  Moore  and  Samuels 
(1931)  came  forward  with  the  demonstration  that  a  few  weeks  of  a 
diet  deficient  in  vitamin  B,  or  a  quantitatively  inadequate  diet  con- 
taining vitamin  B,  caused  in  male  rats  regressive  changes  in  the 
accessory  organs  which,  however,  could  be  counteracted  by  the 
administration  of  testicular  hormone  or  anterior  pituitary  extracts. 
They  concluded  that  the  primary  lesion  due  to  inadequate  feeding 
was  located  in  the  pituitary  gland  and  that  as  a  result  of  the  di- 
minished hypophyseal  activity  the  testes  received  insufficient  gona- 
dotrophic  stimulus  and  were  consequently,  unable  to  produce  the 
male  sex  hormone  required  for  normal  functioning  of  the  accessory 
glands.  A  similar  state  of  'pseudo-hypophysectomy'  was  described 
by  Mulinos  and  Pomerantz  (1941)  in  rats  as  the  result  of  a  diet 
which  was  qualitatively  adequate  but  halved  in  quantity;  further 
supporting  evidence  was  later  provided  by  several  groups  of 
investigators  (Pazos  and  Huggins,  1945;  Goldsmith  and  Nigrelli, 
1950;  Grayhack  and  Scott,  1952).  In  certain  animal  species  sperma- 
togenesis was  also  shown  to  be  affected  by  a  vitamin  B-deficient 

Fructose  and  Fructolysis  149 

diet  (Marrian  and  Parkes,  1928;  Dunn,  Morris  and  Dubnik,  1947; 
Elson  and  Koller,  1948). 

Lutwak-Mann  and  Mann  (1950a,  b,  1951)  applied  chemical 
methods  to  the  study  of  changes  brought  about  in  the  secretory 
function  of  rat  accesory  organs  by  vitamin  B-deficiency  and  inani- 
tion, and  found  that  in  rats  maintained  for  four  weeks  on  a  deficient 
diet  the  content  of  fructose  and  citric  acid  in  the  accessory  glands 
was  reduced  to  a  castrate  level.  By  treatment  with  testosterone  pro- 
pionate (0-2  mg.  daily  for  one  week)  or  with  chorionic  gonadotro- 
phin  (200  units  every  other  day  for  two  weeks),  the  secretory  activity 
of  the  glands  could  be  completely  restored.  A  further  example  of  the 
effect  of  an  unbalanced  diet  on  the  process  of  fructose  secretion  was 
provided  by  Lutwak-Mann  (1951)  who  found  that  a  diet  with  exces- 
sive fat  content,  even  though  not  protein-  or  vitamin-deficient,  also 
caused  regression  in  rat  accessory  organs.  Mann  and  Walton  (1953) 
made  a  study  of  the  effect  of  underfeeding  on  the  genital  functions 
in  the  bull  and  found  that,  in  contrast  to  the  testes,  the  secretory 
function  of  the  male  accessory  glands  was  markedly  affected  by 
underfeeding.  The  concentration  of  fructose  and  citric  acid  in  the 
semen  of  the  underfed  bull  decreased  by  30%  and  60%  respectively, 
of  the  original  levels.  In  the  bull,  however,  unlike  in  the  rat,  the 
effects  of  malnutrition  as  well  as  the  recovery  after  the  animal  has 
been  transferred  back  to  its  normal  diet,  developed  comparatively 

The  enzymic  mechanism  of  fructose  formation 

The  experimental  evidence  available  at  present  brought  out  the 
essential,  though  as  yet  not  fully  understood,  role  of  the  testicular 
hormone  in  the  formation  of  fructose  by  the  secretory  apparatus 
of  the  male  accessory  glands,  and  indicated  that  blood  glucose  is 
the  precursor  of  seminal  fructose.  Further  details  of  the  mechanism 
whereby  glucose  is  converted  in  the  accessory  gland  tissue  to  fruc- 
tose, were  obtained  from  in  vitro  experiments;  these  showed  that 
small  amounts  of  fructose  are  formed  as  a  result  of  incubation  of 
minced  accessory  gland  tissues  with  glucose,  and  that  these  tissues 
possess  the  entire  enzymic  system  which  can  convert  glucose  to 
fructose  (Mann  and  Lutwak-Mann,  1948,  \95\a,  b). 

It  is  an  estabUshed  fact  that  certain  phosphorylated  derivatives  of 

1 50  The  Biochemistry  of  Semen 

fructose,  such  as  6-phosphofructofuranose  (Neuberg  ester)  and  1  :  6- 
diphosphofructofuranose  (Harden- Young  ester),  are  formed  as  in- 
termediary substances  in  the  normal  carbohydrate  metaboUsm  of 
muscle,  liver  and  other  animal  organs.  However,  in  the  majority 
of  animal  tissues  these  phosphofructoses  do  not  yield  free  fructose 
but  are  metabolized  further  to  form  pyruvic  acid  and  lactic  acid. 
In  the  semen,  however,  there  are  present  in  high  concentration 
enzymes  which  belong  to  the  group  of  phosphatases  and  include 
the  'alkaline'  phosphatase;  the  latter  capable  of  splitting  a  number 
of  phosphohexoses,  including  6-phosphofructose,  1-phosphofructose 
and  1  :  6-diphosphofructose,  to  phosphoric  acid  and  free  fructose. 
The  alkaline  phosphatase  found  in  semen  is  derived  from  several 
accessory  organs  of  reproduction  but  its  principal  source  in  higher 
animals  is  the  seminal  vesicle.  Owing  to  this  fact,  the  usual  channels 
of  carbohydrate  metabolism  are  diverted  in  the  vesicular  tissue: 
phosphofructoses  are  not  metabolized  to  lactic  acid,  as  would  be 
the  case  e.g.  in  muscle,  but  are  dephosphorylated  instead,  so  that 
free  fructose  is  formed. 

In  extracts  made  from  bull  seminal  vesicle  tissue  it  is  possible  to 
demonstrate  the  following  reactions.  When  1-phosphoglucose  is 
incubated,  6-phosphoglucose  is  formed  through  the  action  of  phos- 
phoglucomutase;  next,  part  of  6-phosphoglucose  is  converted  by 
phosphohexose  isomerase  into  6-phosphofructose.  The  equilibrium 
mixture  of  the  two  6-phosphohexoses  is  acted  upon  by  phosphatase 
and  yields  a  mixture  of  free  glucose  and  free  fructose.  Phosphohexose 
isomerase,  together  with  alkaline  phosphatase  is  also  present  in  the 
seminal  vesicle  secretion  and  seminal  plasma.  Ram  seminal  plasma 
in  particular,  is  a  rich  source  of  phosphatase  active  towards  phos- 
phorylated  sugars  (Table  17,  p.  120). 

The  fact  that  whereas  a  mixture  of  glucose  and  fructose  is  the 
result  of  the  phosphatase  activity  in  the  glandular  tissue,  yet,  only 
one  sugar,  that  is  fructose,  accumulates  in  the  secretion,  may  have 
its  explanation  in  a  more  effective  re-utilization  of  glucose  than 
fructose,  by  the  glandular  tissue  itself.  Thus,  we  have  found  that 
tissue  slices  from  the  rat  coagulating  gland  can  glycolyse  anaero- 
bically  glucose  at  a  much  higher  rate  than  fructose;  this,  in 
turn,  may  be  due  to  the  ability  of  the  tissue  to  re-phosphorylate 
more  effectively  glucose  to  6-phosphoglucose,  than  fructose   to 

Fructose  and  Fructolysis  151 

6-phosphofructose.  Such  evidence  as  is  at  present  available,  derived 
from  both  in  vivo  and  ///  vitro  experiments,  indicates  that  the 
enzymic  reactions  involved  in  the  conversion  of  blood  glucose  to 
seminal  fructose  are  as  follows. 

Blood  glucose 


^  Phosphorylase 


\  Phosphoglucomutase  Phosphohexose 

6-Phosphoglucose \ >'6-Phosphofructose 

f  \ 

Glucokinase  Alkaline  phosphatase 



Glucose  ^^  Seminal  fructose 

Anaerobic  and  aerobic  utilization  of  carbohydrate  by  spermatozoa 

The  spermatozoa  of  the  sea-urchin  and  certain  other  animals 
derive  their  energy  for  movement  chiefly  from  respiratory  processes; 
in  contrast,  the  survival  and  motility  of  sperm  ejaculated  by  animals 
with  internal  fertilization,  such  as  mammals,  is  possible  for  most 
of  them  also  in  absence  of  oxygen,  provided  that  the  sperm  cells 
remain  in  contact  with  seminal  plasma.  Mammalian  spermatozoa 
possess  only  a  negligible  reserve  of  intracellular  glycogen  and  depend 
therefore,  under  anaerobic  conditions,  on  an  extracellular  source  of 
energy.  In  species  which  contain  fructose  as  a  normal  constituent 
of  the  seminal  plasma,  anaerobic  fructolysis  is  the  metabolic  process 
which  enables  the  spermatozoa  to  survive  without  oxygen.  Should, 
however,  the  spermatozoa  become  separated  from  the  seminal 
plasma  by  centrifugation  and  washing,  they  could  not  carry  on 
anaerobically  unless  the  seminal  plasma  were  restored  or  replaced 
by  glycolysable  carbohydrate. 

The  stimulating  effect  of  pure  sugars  on  sperm  motility  has  been 
noticed  by  some  of  the  early  investigators  of  semen.  In  1931  Ivanov 
observed  that  dog  spermatozoa  suspended  in  an  isotonic  solution  of 
glucose  and  phosphate  retained  their  motility  when  the  respiration 
had  been  abolished  either  by  poisoning  with  cyanide  or  by  replace- 
ment of  oxygen  with  hydrogen.  This  observation  is  of  particular 
interest  in  view  of  the  fact  that  the  dog  has  no  seminal  vesicles  and 

152  The  Biochemistry  of  Semen 

no  fructose  in  the  seminal  plasma;  the  possibility,  of  course,  must 
not  be  overlooked  that  there  may  be  in  dog  semen  some  other  sub- 
stance of  nutrient  value  to  the  spermatozoa.  Redenz  (1933)  has 
shown  that  bull  spermatozoa  glycolyse  glucose,  fructose,  and  man- 
nose  to  lactic  acid,  and  that  the  presence  of  these  sugars,  but  not 
that  of  sucrose,  lactose,  or  glycogen,  is  beneficial  to  sperm  motility. 
His  findings  were  confirmed  by  others  and  it  has  since  become 
an  established  fact  that  the  metabolism  of  spermatozoa  in  several 
mammalian  species  including  man,  ram  and  bull,  is  predominantly 
of  a  glycolytic  character  (Ivanov,  1935;  Shergin,  1937;  Comstock, 
1939;  MacLeod,  1939,  19436;  Lardy  and  Phillips,  \9A\a\  Moore  and 
Mayer,  1941;  Henle  and  Zittle,  1942;  Ross,  Miller  and  Kurzrok, 
1941;  Salisbury,  1946). 

Sperrnatozoa  obtained  directly  from  the  epididymis  of  a  bull, 
ram,  or  boar,  resemble  washed  ejaculated  sperm  in  that  they  are 
incapable  of  survival  under  purely  anaerobic  conditions.  While  in 
the  epididymis,  the  spermatozoa  have  no  access  to  fructose  and  are 
immotile;  the  onset  of  motiUty  coincides  with  their  passage  along 
the  male  genital  tract  and  contact  with  the  seminal  plasma.  The  acti- 
vating influence  of  fructose  on  previously  immotile  spermatozoa 
can  be  convincingly  demonstrated  in  a  simple  manner.  Fresh  epi- 
didymal  spermatozoa  are  suspended  in  bicarbonate-Ringer  solution; 
two  'hanging  drops',  one  of  the  suspension  and  another,  a  little 
further  away,  containing  a  1  %  solution  of  fructose  in  bicarbonate- 
Ringer  solution,  are  placed  on  the  underside  of  a  cover-sUp;  to 
observe  the  motility  of  the  sperm  under  the  microscope,  the  cover- 
slip  is  fixed  to  the  top  of  a  small  glass  chamber  in  which  one  can 
create  anaerobic  or  aerobic  conditions  by  passing  through  the  cham- 
ber a  gas  mixture  of  95%  N2-5%  CO2  or  95%  02-5%  CO2.  In  the 
absence  of  oxygen,  the  spermatozoa  can  be  seen  to  be  almost  com- 
pletely immotile,  but  when  the  two  drops  are  brought  together  the 
sperm  movement  begins  and  continues  for  a  long  time.  Aerobically, 
the  effect  of  fructose  is  less  striking  because  oxygen  induces  endo- 
genous respiration  and  this  in  itself  provokes  motility  in  epididymal 
spermatozoa.  However,  even  in  the  presence  of  oxygen,  fructose 
still  has  some  influence  owing  to  the  process  of  aerobic  fructolysis. 

Under  anaerobic  conditions,  the  final  product  of  sperm  fructolysis, 
lactic  acid,  cannot  be  oxidized  further.  In  the  presence  of  oxygen, 

Fructose  and  Fructofysis  153 

however,  the  situation  differs  in  that  the  rate  of  fructose  utiliza- 
tion becomes  smaller,  and  moreover,  lactic  acid  undergoes  further 
oxidation,  thus  providing  an  additional  source  of  metabolic  energy. 
It  remains  for  further  study  to  ascertain  what  type  of  carbohydrate 
metabolism  predominates  in  spermatozoa  during  their  existence  in 
either  the  male  or  female  genital  tract.  However,  so  far  as  in  vitro 
studies  are  concerned,  they  show  that  lactic  acid  can  be  efficiently 
oxidized  by  spermatozoa  even  when  the  partial  pressure  of  oxygen 
has  been  reduced  to  a  level  as  low  as  that  which  normally  prevails 
in  animal  tissues  (Mann,  1951Z));  suitably  diluted  suspensions  of  ram 
spermatozoa  show  in  presence  of  1  %  O2  a  respiratory  rate  as  high 
as  in  air,  and  lactate  is  capable  of  maintaining  the  oxygen  uptake 
equally  well  in  1%  as  in  20%,  oxygen. 

Pasteur  effect  and  the  ^metabolic  regulator'' 

It  was  said  earher  that  the  spermatozoa  obtained  directly  from 
the  epididymis  in  some  ways  behave  like  suspensions  of  washed 
ejaculated  sperm;  as  a  matter  of  fact,  however,  these  two  types  of 
sperm  cells  possess  distinct  characteristics  (Henle  and  Zittle,  1942; 
Lardy,  Hansen  and  Phillips,  1945).  Washed  epididymal  bovine 
spermatozoa  have  a  lower  endogenous  respiration  than  those  in 
ejaculated  bull  semen.  But  if  sugar  is  added  or  if  the  spermatozoa 
are  removed  from  the  epididymis  after  a  period  of  storage  in  the 
refrigerator,  then  their  oxygen  uptake  is  distinctly  higher.  More- 
over, on  addition  of  sugar,  epididymal  spermatozoa  produce  lactic 
acid  much  more  rapidly  under  anaerobic  than  aerobic  conditions, 
whereas  in  ejaculated  sperm  the  rate  of  glycolysis  is  not  much 
higher  in  the  presence  than  in  the  absence  of  oxygen.  To  account 
for  these  differences,  the  Wisconsin  workers  determined  the  rate  of 
anaerobic  and  aerobic  glycolysis  as  well  as  of  oxygen  uptake,  in 
epididymal  spermatozoa  to  which  glucose  was  added,  and  calcu- 
lated the  'Meyerhof  oxidation  quotient'  which  measures  the  Pasteur 
effect,  that  is  the  extent  to  which  glycolysis  is  inhibited  by  oxygen. 
The  average  value  for  the  Meyerhof  quotient  calculated  from  twelve 
experiments  on  bull  epididymal  sperm  was  9-6,  as  against  5  recorded 
for  ejaculated  sperm  (Lardy,  1952).  This  difference,  according  to 
Lardy,  Ghosh  and  Plant  (1949),  is  due  to  the  presence  in  bull  sper- 
matozoa of  a  'metabolic  regulator'  which  occurs  in  the  epididymal 

1 54  The  Biochemistry  of  Semen 

sperm  in  a  'bound  form'  but  is  released  in  an  'active  form'  after 
ejaculation.  Continuing  their  study,  these  workers  observed  that 
heat-inactivated  bull  semen,  or  semen  and  testicular  extracts  heated 
with  sodium  hydroxide,  increased  the  rate  of  aerobic  fermentation 
of  sugars  by  baker's  yeast,  without  affecting  markedly  yeast  respi- 
ration or  anaerobic  fermentation.  The  yeast-stimulating  factor  was 
extracted  with  carbon  tetrachloride  from  alkaline  hydrolysates  of 
hog  testes  and  obtained  in  the  form  of  yellow  coloured  crystals 
which  proved  to  be  elementary  sulphur  (Ghosh  and  Lardy,  1952). 
Yeast  reduces  sulphur  to  HgS  which  is  probably  the  agent  ultimately 
responsible  for  the  stimulation  of  the  aerobic  fermentation.  The 
identity  of  the  yeast  factor  with  sulphur  was  verified  by  reproducing 
the  stimulating  effect  on  yeast  with  pure  rhombic  sulphur.  Sulphur 
as  such,  however,  cannot  be  the  sperm  'regulator'  since  it  is  without 
influence  on  the  Pasteur  effect  in  epididymal  spermatozoa.  On  the 
other  hand,  a  number  of  sulphydryl  compounds  such  as  cysteine, 
reduced  glutathione  and  hydrogen  sulphide  have  been  found  to 
stimulate  the  respiration  and  aerobic  glycolysis  of  epididymal 
sperm  and  the  possibility  remains,  that  the  'metabolic  regulator' 
is,  in  fact,  a  sulphydryl  compound,  liberated  during  ejaculation 
from  the  spermatozoa,  with  a  sulphydryl  group  in  labile  form,  which 
can  be  easily  removed  and  oxidized  to  sulphur  by  alkaline  hydrolysis. 
The  peculiar  changes  in  the  metabolic  properties  of  spermatozoa 
during  cold-storage  of  the  epididymis,  are  equally  in  need  of  eluci- 
dation. A  problem  which  also  deserves  further  biochemical  study 
is  the  'ripening'  phenomenon  which  takes  place  in  the  spermatozoa 
while  they  remain  in  the  epididymis.  Presumably,  the  metabolism 
of  sperm  in  the  epididymis  is  related  in  some  as  yet  unknown 
manner  to  the  structural  changes  associated  with  sperm  maturation 
processes,  such  as  the  migration  of  the  'kinoplasmic  droplet'. 

Intermediary  reactions  in  sperm  fructo  lysis  and  the  role  of  phosphorus- 
containing  coenzymes 
The  ability  of  washed  spermatozoa  to  convert  into  lactic  acid 
equally  well  added  fructose,  glucose  and  mannose  is  due  in  all 
probability  to  the  fact  that  the  metabolic  degradation  of  these 
three  sugars  is  initiated  by  the  sam.e  hexokinase-catalysed  reaction 
with  adenosine  triphosphate. 

Fructose  and  Fructolysis  155 

Adenosine  triphosphate  (ATP,  formula  in  Fig.  15)  represents  an 
intracellular  constituent  and  a  coenzyme  of  considerable  importance 
in  the  economy  of  the  sperm  cell.  An  observation  that  a  considerable 
proportion  of  the  acid-soluble  phosphorus  in  bull  spermatozoa 
yields  orthophosphate  after  7  min.  hydrolysis  with  n-HCI  first 
suggested  the  presence  of  ATP  (Lardy  and  Phillips,  1945).  In  the 
same  year,  the  readily-hydrolysable  phosphorus  compound  was 
isolated  from  ram  spermatozoa  and  its  identity  with  ATP  estab- 
lished by  chemical  analysis  (Mann,  1945«,  c);  the  content  of  ATP 
in  ram  spermatozoa  is  2-6-6-6  mg.  labile  phosphorus  or  0-6-1 -5  mg. 
of  adenine  amino-nitrogen  per  100  ml.  semen  (see  also  Table  16). 
The  occurrence  of  ATP  in  ram  and  boar  spermatozoa  has  also  been 
confirmed  by  Ivanov,  Kassavina  and  Fomenko  (1946)  who  found 
that  the  phosphorus  compound  which  they  purified  from  sperm 
induced  contractions  of  muscle  actomyosin  threads  in  the  same 
manner  as  ATP  isolated  from  skeletal  muscle.  ATP  was  also  found 
in  sea-urchin  spermatozoa  (Rothschild  and  Mann,  1950),  the  con- 
centration of  ATP  in  the  semen  of  Echinus  escidentus  resembling 
that  found  in  the  ram. 

Spermatozoa,  even  after  they  have  been  repeatedly  washed  so  as 
to  remove  the  phosphatases  present  in  seminal  plasma,  continue  to 
exhibit  a  high  phosphatase  activity  against  ATP,  and  all  evidence 
available  at  present  points  to  sperm  ATP-ase  as  the  enzyme  which 
is  directly  responsible  for  the  supply  of  energy  essential  for  normal 
motihty  and  survival  of  the  sperm  cell.  The  losses  due  to  utilization 
of  ATP  are  made  good  by  re-synthesis  which  takes  place  during  the 
normal  metabolism  of  spermatozoa,  and  any  interference  with  inter- 
mediary enzymic  reactions  which  renders  the  sperm  cell  incapable 
of  breaking  down  or  building  up  ATP,  leads  to  a  decrease  in  both 
metabolism  and  motility.  Using  ram  spermatozoa  as  experimental 
material  under  a  variety  of  conditions,  we  have  found  that  a  diminu- 
tion in  the  content  of  ATP  invariably  coincides  with  impaired  sperm 
motility  (Mann,  1945^7,  b,  c).  Thus,  for  instance,  in  ram  spermatozoa 
deprived  of  the  fructose-containing  seminal  plasma  by  washing, 
ATP  content  as  well  as  motility  went  down  simultaneously  on 
anaerobic  incubation,  but  both  ATP  and  motility  could  be 
preserved  anaerobically  in  sperm  suspensions  provided  with 
glycoly sable  material. 


The  Biochemistry  of  Semen 

The  activity  of  hexokinase,  the  enzyme  which  brings  about  the 
initial  reaction  between  ATP  and  glycolysable  sugar,  can  be  demon- 
strated directly  in  washed  spermatozoa  (Mann,  1945/)).  If  we  add 
to  a  ram  sperm  suspension  sugar  (glucose,  fructose  or  mannose), 



Spermatozoa                  Seminal    Plasma 

rifc— ^ 



1        A 



Diphosphofructose          "J 

1     "li^"" 

^l'                           OH>— f  OH 


Phosphotrjose             i^     ^ 


1                  D  (-^Fructose 

^H^K    ^^ 


1         ^N          A 




Lactic  acid 

AXR       > 


^            .CHoCHCOOH 

Phosphopyruvic        "^  ^^ 

acid                         y 



-< i 

Pyruvic  acid 

L                        J 

Fig.  15.  Diagrammatic  representation  offructolysis  in  semen. 

sodium  fluoride,  and  ATP,  there  is  on  incubation  a  rapid  disap- 
pearance of  half  of  the  readily-hydrolysable  phosphorus  of  ATP 
and  formation  of  adenosinediphosphate  (ADP)  and  6-phospho- 
hexose.  Of  course,  without  added  ATP  and  fluoride,  6-phospho- 
hexose  does  not  accumulate  but  the  degradation  of  sugar  continues 
uninterruptedly  to  its  final  stage,  i.e.  the  formation  of  lactic  acid. 

Fructose  and  Fructolysis  1 57 

If  either  glucose  -alone  or  fructose  alone  is  used  as  substrate,  the 
rate  of  lactic  acid  production  is  the  same  (Mann  and  Lutwak-Mann, 
1948),  but  if  washed  spermatozoa  are  made  to  act  on  a  1  :  1  mixture 
of  glucose  and  fructose,  then  the  rate  of  fructose  utihzation  becomes 
much  less  than  50%  (Mann,  \95\b).  This  'sparing  effect'  of  glucose 
on  the  utilization  of  fructose  is  probably  due  to  the  competitive 
inhibition  of  sperm  hexokinase.  Slein,  Cori  and  Cori  (1950)  have 
shown  that  when  brain  or  yeast  hexokinase  acts  upon  ATP  and  on 
an  equimolar  mixture  of  glucose  and  fructose,  the  aldosugar  is  phos- 
phorylated  much  more  rapidly  than  the  ketosugar.  Under  natural 
conditions  only  fructose  is  present  in  whole  semen,  but  not  glucose, 
so  that  the  possibility  of  the  latter  interfering  with  fructolysis  does 
not  arise.  But  there  is  preferential  utilization  of  glucose  in  the  acces- 
sory glands,  directly  responsible  for  the  accumulation  of  fructose, 
and  this  may  be  due  to  a  stronger  affinity  of  hexokinase  for  glucose 
than  for  fructose.  A  competition  for  hexokinase,  between  glucose 
and  fructose,  is  also  consistent  with  the  observation  that  in  bull 
semen  incubated  with  an  egg-yolk-diluent,  the  initial  rate  of  fructo- 
lysis is  temporarily  retarded  (Vantienhoven,  Salisbury,  VanDemark 
and  Hansen,  1952),  as  is  also  the  case  in  semen  incubated  with  cow 
follicular  fluid  (Lutwak-Mann,  1954);  both  egg-yolk  and  follicular 
fluid  contain  glucose. 

The  phosphohexose  formed  from  fructose  as  a  result  of  hexo- 
kinase activity  is  6-phosphofructofuranose;  in  the  case  of  glucose 
the  product  is  6-phosphoglucopyranose.  The  latter,  however,  is 
readily  converted  to  6-phosphofructose  by  phosphohexose  iso- 
merase,  and  from  this  stage  onwards,  the  enzymic  degradation  of 
glucose  and  fructose  is  identical.  The  chain  of  events  which  in  whole 
semen  leads  from  fructose  to  lactic  acid,  is  diagrammatically  depicted 
in  Fig.  15.  In  the  normal  course  of  fructolysis,  6-phosphofructose  is 
phosphorylated  by  ATP  in  a  reaction  catalysed  by  phosphofruc- 
tokinase,  to  yield  1  :  6-diphosphofructose  and  ADP;  disphospho- 
fructose  is  next  split  by  zymohexase  into  two  molecules  of  phospho- 
triose.  Like  the  action  of  phosphofructokinase,  that  of  zymohexase 
was  demonstrated  directly  in  spermatozoa  (Mann,  1945^). 

The  subsequent  steps  in  sperm  fructolysis  are  analogous  to  the 
corresponding  phases  in  muscle  glycogenolysis  and  blood  glucolysis, 
and   involve   the   participation   of  cozymase   (diphosphopyridine 

158  The  Biochemistry  of  Semen 

nucleotide)  which  as  Winberg  (1941)  showed,  is  a  characteristic 
intracellular  constituent  of  spermatozoa.  The  cozymase-catalysed 
phase  of  fructolysis  consists  of  two  closely  interwoven  oxidoreduc- 
tion  processes  (Mann,  19456;  Mann  and  Lutwak-Mann,  1947). 


_,,  ,     ^  .  (,    Phosphotriose  dehydrogenase    )         t^,  i        ,  •         •  j 

Phosphotriose  — ^^ . ^-^  Phosphoglyceric  acid 


H2  NaF  inhibits 

\  Y 

Cozymase  Phosphopyruvic  acid 


Ha  cozymase 


Lactic  acid^ ; Pyruvic  acid 

Lactic  dehydrogenase  -^ 

The  first  oxidoreduction  involves  the  oxidation  of  phosphotriose 
to  phosphoglyceric  acid  by  phosphotriose  dehydrogenase,  and  a 
simultaneous  reduction  of  cozymase  to  dihydrocozymase;  the  oxida- 
tion of  phosphotriose  is  coupled  with  an  esterification  of  inorganic 
phosphate  and  the  synthesis  of  ATP;  the  oxidation  product,  phos- 
phoglyceric acid,  is  converted  by  enolase  to  phosphopyruvic  acid, 
the  phosphate  of  which  is  transferred  to  ADP,  thus  producing 
pyruvic  acid  and  ATP.  The  second  oxidoreduction  is  between 
dihydrocozymase  and  pyruvic  acid:  dihydrocozymase  is  oxidized  to 
cozymase,  and  pyruvic  acid  is  reduced  by  lactic  dehydrogenase  to 
L(+)-lactic  acid.  When  washed  ram  spermatozoa  are  treated  with 
fluoride  (to  inhibit  enolase),  and  incubated  with  added  phospho- 
triose and  pyruvate,  the  two  oxidoreduction  processes  continue  as 
usual  but  in  addition  to  lactic  acid  there  is  an  accumulation  of 
phosphoglyceric  acid.  lodoacetate,  on  the  other  hand,  abolishes  the 
oxidoreductions  in  washed  sperm  and  thus  deprives  them  of  the 
ability  to  produce  lactic  acid. 

The  not  unimpressive  array  of  facts  available  from  the  outlined 
studies  on  spermatozoa  strengthens  the  belief  that  ATP  is  the  car- 
dinal link  between  the  activity  of  spermatozoa  on  the  one  hand, 
and  carbohydrate  metabolism  on  the  other.  In  whole  semen,  ATP 
acts  continually  as  phosphate-donor  and  acceptor  in  the  course  of 
fructolysis.  The  content  of  ATP  and  with  it  the  motility  of  ejaculated 

Fructose  and  Fructolysis  1 59 

spermatozoa,  both  depend  on  the  maintenance  of  the  normal 
metaboHsm  of  fructose. 

We  still  remain  confronted  with  two  questions  to  which,  it  is 
confidently  hoped,  further  research  will  bring  answers.  One  involves 
the  as  yet  obscure  position  in  the  semen  of  animal  species  which  lack 
fructose.  Secondly,  one  wonders  why  nature  should  have  chosen 
fructose  and  not  glucose,  as  the  natural  substrate  for  sperm  meta- 
bolism. At  this  point,  conditions  in  another  body  fluid,  the  milk, 
come  to  mind;  there,  the  occurrence  of  lactose  poses  a  somewhat 
similar  question.  But  in  considering  the  matter,  several  facts  must 
be  taken  into  account.  To  begin  with,  if  glucose  and  not  fructose 
were  present  in  semen,  its  concentration  could  hardly  be  expected 
to  exceed  that  of  blood  and  other  body  fluids.  Thus,  it  might  not 
be  sufficient  to  satisfy  the  metabolic  requirements  of  spermatozoa 
which,  unlike  most  other  animal  cells,  are  capable  of  utilizing  fruc- 
tose anaerobically;  it  is  worth  noting  that  on  the  whole,  yeasts  and 
bacteria  are  also  unable  to  consume  fructose  at  the  same  rate  as 
glucose.  Presumably,  this  enables  the  spermatozoa  to  draw  freely 
upon  seminal  fructose  without,  as  it  were,  competition  from  other 
tissues.  Lastly,  the  intimate  relationship  between  seminal  fructose 
and  the  male  sex  hormone  must  not  be  lost  sight  of;  it  would  be 
rather  difficult  to  envisage  a  similar  dependence  in  the  case  of 
glucose,  bearing  in  mind  the  ubiquitous  occurrence  and  physio- 
logical function  of  this  sugar. 


Spermine,  Choline,  Ergothioneine ,  and 
certain  other  Bases  in  Semen 

Spermine.  Occurrence  of  crystalline  spermine  in  human  semen;  its 
chemical  nature  and  properties.  Derivatives  of  spermine  and  their  use 
in  forensic  medicine.  Synthesis  of  sperrnine.  Spermidine.  Oxidation  of 
spermine  and  spermidine  by  diamine  oxidase.  State  of  spermine  in  semen. 

Choline.  The  Florence  reaction  in  semen.  Enzymic  liberation  of  choline 
from  precursors  in  semen.  Phosphorylcholine  and  glycerylphosphoryl- 
choHne.  Physiological  function  of  free  and  bound  choline.  Choline 

Ergothioneine.  Isolation  of  ergothioneine  from  the  boar  seminal  vesicle 
secretion.  The  function  of  seminal  ergothioneine  and  its  behaviour  towards 
sulphydryl-binding  substances.  Biogenesis  of  ergothioneine. 

Creatine  and  creatinine.  Occurrence  in  mammalian  semen,  and  in  the 
sperm  and  gonads  of  invertebrates.  Phosphocreatine  and  phosphoarginine. 

Adrenaline  and  noradrenaline .  Occurrence  in  semen  and  accessory  organs. 
Enzymic  oxidation.  Pharmacodynamic  properties. 

Among  the  chemical  characteristics  which  distinguish  semen  from 
other  tissues  and  body  fluids  is  the  occurrence  of  certain  nitrogenous 
bases,  largely  betaines,  which  are  rarely  found  elsewhere  in  the 
animal  body.  Of  these,  spermine  is  the  oldest-known,  and  ergo- 
thioneine the  most  recently  discovered. 


Occurrence  of  crystalline  spermine;  its  chemical  nature  and  properties 
When  human  semen  'had  stood  a  little  while,  some  three-sided 
bodies  were  seen  in  it,  terminating  at  either  end  in  a  point;  some 
were  of  the  length  of  the  smallest  grain  of  sand,  and  some  were  a 
little  bigger,  as  in  Fig.  A.  They  were  further  as  bright  and  clear  as  if 
they  had  been  crystals.'  Thus,  in  a  letter  of  November  1677,  addressed 
to  the  Royal  Society,  Antoni  van  Leeuwenhoek  reported  the  dis- 
covery of  the  crystalline  substance  in  semen  which  later  became 



ditld  materia pauallumtempoTiiJieterAt^  in  ea,  ohjtrvabsrjtur  tri- 
idteraiesfi^ur^  ^b  utraque  pirte  tn  acttieum  defin'r:tis  ^  quthuf- 
dim  icngitudfi  minuttfiiwd'  arena^  aliqu£  ^ltqt4ihtH  urn  wajores^ 
A  f^  l\  %    «^  fij*  A.  Frateredy  adeo  ntttd£  &o  ptUucida^   acfi 

"    i|  V  ^'     cr\fiallin£  juijjent. 


Crystals  in  human  semen  as  seen  (from  top  to  bottom)  by  Leeuwenhoek 
(1677),  Fuerbringer  (1881)  and  Poehl  (1898). 

Spermine,  Choline,  Ergothioneine  161 

known  as  spermine.  This  was  actually  the  letter  in  which  Leeuwen- 
hoek  also  communicated  for  the  first  time  the  discovery  of  living 
spermatozoa  and  their  movement  in  fresh  semen;  it  was  published 
the  following  year  in  the  Philosophical  Transactions  (Plate  VII). 
During  the  200  years  which  followed,  the  same  crystalline  substance 
was  rediscovered  by  several  investigators,  most  of  whom  were 
apparently  unaware  of  either  Leeuwenhoek's  original,  or  of  the 
others'  later  observations. 

Vauquelin(1791)  observed  in  a  semen  sample  which  he  left  standing 
for  four  days,  the  deposition  of  'cristaux  transparens,  d'environ 
une  ligned  de  long,  tres-minces,  &  qui  se  croisent  souvent  de  maniere 
a  representer  les  rayons  d'une  roue.  Ces  cristaux  isoles  nous  ont 
offert,  a  Faide  d'un  verre  grossissant,  la  forme  d'un  solide  a  quatre 
pans,  termines  par  des  pyramides  tres-allongees,  a  quatre  faces.' 
After  having  studied  the  properties  and  behaviour  of  these  crystals 
towards  different  solvents,  Vauquelin  concluded  that  'la  nature  de 
ces  cristaux  est  analogue  a  celle  du  phosphate  de  chaux  ou  la  bas 
des  os'.  The  belief  that  the  sperm  crystals  consist  of  ordinary  phos- 
phate persisted  throughout  the  best  part  of  the  next  century.  In  the 
meantime,  however,  the  same  crystalline  substance  was  found  out- 
side the  semen  in  other  tissues  and  body  fluids,  including  sputum, 
leucaemic  blood,  liver,  spleen  and  old  pathological-anatomical  pre- 
parations, so  that  towards  the  close  of  the  XlXth  century  spermine 
was  already  known  by  no  less  than  ten  names  of  various  distin- 
guished clinicians,  anatomists  and  physiologists,  including,  in 
chronological  order,  Charcot,  1853;  Foerster,  1859;  Harting,  1859; 
White,  1861  ('leucosine');  Friedrich,  1864;  Huppert,  1864;  Boettcher, 
1865;  Neumann,  1866;  Eberth,  1869;  Ley  den,  1872;  and  Zenker, 
1876.  But  in  the  end,  the  medical  world  at  large  restricted  itself 
largely  to  the  use  of  two  names,  'Charcot-Leyden  crystals'  with 
reference  to  organs  and  sputum  ('asthma  crystals'),  and  'Boettcher 
crystals'  with  reference  to  semen.  Boettcher  himself  preferred  to 
call  the  substance  'Spermatin',  and  regarded  it  as  a  protein;  he 
published  his  paper  'Farblose  Krystalle  eines  eiweissartigen  Korpers 
aus  dem  menschlichen  Sperma  dargestellt'  in  1865,  without  however, 
taking  the  trouble  to  mention  the  previous  investigators. 

The  credit  for  having  been  the  first  to  recognize  spermine  as  the 
phosphate  of  a  new  organic  base,  is  due  to  Schreiner  (1878)  who 

162  The  Biochemistry  of  Semen 

succeeded  in  preparing  a  number  of  derivatives  of  spermine  includ- 
ing the  hydrochloride,  but  who  unfortunately  deduced  from  his 
analyses  the  wrong  formula  for  the  base,  C2H5N.  In  consequence  of 
this,  spermine  was  confused  with  ethyleneamine,  CoH^NH,  and  with 
piperazine.  For  years  to  follow,  piperazine  was  offered  by  a  large 
pharmaceutical  firm  in  Berlin  under  the  trade  name  of  'Spermin', 
and  as  late  as  1903  the  formula  of  piperazine  appeared  under  the 
name  of  spermine  in  Thierfelder's  Hoppe-Seyler' s  Handbuch  der 
chemischen  Analyse.  A  great  advocate  of  the  manifold  curative  pro- 
perties of  'real'  spermine,  i.e.  as  isolated  from  human  semen,  bull 
testes,  or  other  organs,  was  Alexander  von  Poehl,  who  believed  in  the 
'action  of  spermine  as  a  physiological  tonic  on  auto-intoxications' 
(1893),  and  who  is  best  known  for  the  monograph  Die physiologisch- 
chemischen  Grundlagen  der  Spermintheorie  which  he  published  in 
St.  Petersburg  in  1898.  Poehl's  book  contains  the  records  of  numer- 
ous cases  ranging  from  scurvy  to  syphilis,  treated,  apparently  suc- 
cessfully, with  the  'Sperminum  Poehl'.  His  pharmacological  and 
clinical  work  aroused  much  controversy,  was  subjected  to  severe 
criticism,  and  was  finally  altogether  rejected.  Yet,  it  is  not  entirely 
improbable  that  there  is  some  justification  for  Poehl's  'spermine 
theory'.  Apart  from  its  general  pharmacodynamic  properties  similar 
to  those  of  other  biological  polyamines  (Guggenheim,  1940),  sper- 
mine may  well  possess  some  other,  more  specific  pharmacological 
activity.  Administered  parenterally,  spermine  is  known  to  be 
toxic  to  mice,  rats  and  rabbits  (Rosenthal,  Fisher  and  Stohlman, 
1952).  It  has  also  been  shown  to  possess  bacteriostatic  properties. 
The  inhibition  of  the  growth  of  Staphylococcus  aureus  by  human 
seminal  plasma  can  be  attributed,  according  to  Gurevitch  and  his 
colleagues  (1951),  to  the  high  content  of  spermine  in  human  semen. 
Another  striking  example  of  the  growth-inhibiting  action  of  sper- 
mine has  been  provided  by  Hirsch  and  Dubos  (1952);  following  up 
an  observation  that  the  extraction  of  animal  tissues  with  mixtures  of 
water  and  ethanol  yields  material  with  tuberculostatic  activity  in 
vitro,  these  authors  isolated  from  tissue  extracts  a  crystalline  anti- 
mycobacterial  substance  which  they  found  to  be  identical  with 
spermine  phosphate. 

Leaving  aside  Poehl's  pharmacological  observations,  one  must 
nevertheless  appreciate  his  contribution  to  the  chemistry  of  spermine. 

Spermine,  Choline,  Ergothioneine  163 

Not  only  was  he  able  to  refute  the  mistaken  belief  in  the  identity 
of  spermine  and  diethylenediamine  but  he  was  also  the  first 
to  analyse  correctly  the  gold  salt  and  the  chloroplatinate  of  sper- 
mine and  to  establish  that  'the  organic  base  which  is  at  the  bottom 
of  these  double  salts  would  have  the  composition,  C5H14N2  (1898, 

However,  not  until  1924  was  conclusive  chemical  and  crystallo- 
graphic  evidence  brought  forward  to  prove  the  identity  of  spermine 
isolated  from  semen  with  the  base  obtained  by  similar  methods  from 
various  animal  organs  and  also  from  yeast.  Credit  for  this  is  due  to 
Otto  Rosenheim  (1924).  In  1924,  Dudley,  Mary  Rosenheim  and  O. 
Rosenheim  in  England,  and  Wrede  in  Germany,  concluded  from 
the  molecular  weight  estimations  of  benzoylspermine  and  m-mixo- 
benzoylspermine,  respectively,  that  the  molecular  formula  of  sper- 
mine is  C10H26N4,  and  not  as  formerly  assumed  C5H14N2. 

Rosenheim  and  his  colleagues  obtained  spermine  in  the  free  state 
as  a  crystalline,  optically  inactive  substance  which  melts  between 
55°  and  60°,  and  distills  at  about  150°  in  vacuo  without  decomposi- 
tion. They  also  found  that  the  base  is  stable  in  hot  concentrated 
alkali  and  in  boiling  hydrochloric  acid.  Their  relatively  simple 
method  of  isolation  depends  on  the  steam-distillation  of  spermine 
from  a  strongly  alkaline  solution. 

Derivatives  of  spermine  and  their  use  in  forensic  medicine 

There  are  several  well-defined  compounds  of  spermine,  a  list  of 
which  is  given  in  Table  22.  Apart  from  the  highly  characteristic 
insoluble  phosphate,  spermine  can  be  identified  particularly  easily 
as  a  picrate,  which  can  be  prepared  from  the  free  base,  the  phos- 
phate, or  directly  from  semen.  Spermine  picrate,  like  the  phosphate, 
is  extremely  insoluble  in  water.  Crystallographic  analysis  has  shown 
that  it  is  identical  with  the  substance  responsible  for  the  so-called 
Barberio  reaction,  a  chemical  test  of  diagnostic  value  in  forensic 
medicine.  Barberio's  (1905)  test  consists  in  the  addition  to  semen, 
or  to  an  aqueous  extract  from  the  seminal  stain,  of  picric  acid  in 
concentrated  aqueous  or  ethanolic  solution;  in  the  presence  of  picric 
acid,  there  follows  within  a  few  minutes,  the  formation  of  abundant 
yellow  crystals,  resembling  in  shape  the  crystals  of  spermine  phos- 
phate. The  statement  by  Barberio  that  the  reaction  appears  to  be 


The  Biochemistry  of  Semen 

specific  for  human  semen  as  distinct  from  animal  semen,  has  been 
corroborated  by  other  ItaUan  investigators,  particularly  by  Baecchi 
(1912).  In  the  experience  of  Littlejohn  and  Pirie  (1908),  Barberio's 
reaction  is  best  carried  out  as  follows;  a  small  piece  of  the  stained 
fabric  is  placed  upon  a  glass  slide  and  macerated  in  a  drop  or  two 

Table  22.  Chemical  properties  of  spermine  and  its  derivatives 

Compound  Formula 

Free  base     C10H26N4 

Phosphate    CioH26N4-2H3P04-6H20 

Hydro-         CioH2eN4-4HC] 


Picrate  CioH26N4-4C6H307Na 

Chloro-         CioH26N4-4HCl-4AuCls 

Chloro-         CioH26N4-2H2PtCl6 


Benzoyl        CioHaeNi^COCeHs 

Tetra-  CioH26N4-4CioH608N2S 



Needle-shaped,  colourless,  odour- 
less crystals;  easily  soluble  in 
water,  ethanol  and  butanol,  in- 
soluble in  ether,  benzene  and 
ligroin,  m.p.  55-60°,  m.w.  202 

Lenticular  crystals  from  water, 
long  needles  from  ethanol.  Insol. 
in  cold  water,  sol.  1  :  100  in  boil- 
ing water,  easily  soluble  in  dilute 
acid  or  alkali,  m.p.  240°,  m.w. 
504.  Identical  with  'Boettcher's 
crystals'  and  'Charcot-Leyden 

Short  prismatic  needles,  extremely 
soluble  in  water,  insol.  in 
acetone,  ether  and  chloroform, 
m.p.  300-310° 

Yellow  needles,  become  black  at 
242°,  melt  sharply  with  decom- 
position at  248-250° 

Golden-yellow,  lustrous  leaflets, 
m.p.  225°  (Wrede  218°) 

Orange-yellow,  well-formed  cry- 
stals, m.p.  242-245° 

Lemon-yellow  crystals,  m.p.  288- 

Crystallizes  from  a  solution  in  hot 
acetone  on  addition  of  ligroin,  in 
woolly  balls  of  fine  needles,  m.p. 
155°,  m.w.  618 

CrystalliLes  if  flavianic  acid  is  used 
in  excess.  On  recrystallization 
from  water  the  diflavianate  is 

Spermine,  Choline,  Ergothioneine  165 

of  distilled  water;  to  the  extract  thus  obtained  (concentrated  by 
evaporation,  if  necessary),  a  very  small  drop  of  an  aqueous  saturated 
solution  of  picric  acid  is  added,  by  means  of  a  platinum  loop;  after 
a  minute  or  two  a  cover  slip  is  applied,  and  the  preparation  examined 
under  the  microscope;  'when  fully  developed,  the  crystals  have  the 
form  either  of  obtuse  or  sharp-ended  needles,  or  of  rhombic  prisms 
frequently  crossed  by  a  refrangent  line  at  their  equator.  Sometimes 
crosses  are  formed,  and  more  rarely  stars.'  (For  further  particulars 
concerning  Barberio's  test  see  Harrison,  1932.)  Another  deriva- 
tive of  spermine  used  in  medico-legal  laboratories  is  spermine 
flavianate;  this  crystalline  compound  forms  the  basis  of  Puranen's 
reaction  (Puranen,  1936;  Berg,  1948). 

Synthesis  of  spermine 

The  final  elucidation  of  the  chemical  structure  of  spermine  was 
achieved  in  1926.  The  existence  of  two  chains  A^ — C — C — C — A'^ 
and  of  one  chain  N—C — C — C — C — A^  in  the  spermine  molecule 
was  inferred  by  Dudley,  Rosenheim  and  Starling  from  the  identi- 
fication of  pyrrolidine  and  tetramethyltrimethylene  diamine  as 
degradation  products  of  spermine  hydrochloride  and  decamethyl 
spermine,  respectively.  In  Wrede's  laboratory,  the  presence  of  two 
3C  chains  and  one  AC  chain  was  established  the  same  year  as  a 
result  of  studies  on  split  products  obtained  from  spermine  by  oxidation 
with  molecular  oxygen  in  presence  of  copper.  The  final  proof  was 
provided  by  the  English  investigators  when  they  accomplished  the 
synthesis  of  spermine  and  showed  it  to  be,  a,(5-bis  [y'-amino- 


I  ■  I  II 

NH2         NHCH2CH0  CH2CH2  NH  NH2 

The  oxidation  of  spermine  gives  rise  to  a  volatile  base  associated 
with  the  characteristic  odour  of  semen.  The  appearance  of  the 
semen-like  odour  during  treatment  of  spermine  chloroaurate  with 
metallic  magnesium,  first  described  by  Schreiner  (1878),  probably 
involves  also  an  oxidation.  The  base  is  volatile  in  steam,  and  forms 
a  crystalline  hydrochloride  and  chloroaurate  (m.p.  204-206°);  it  is 
probably  identical  with  A^-y-aminopropylpyrroline. 

166  The  Biochemistry  of  Semen 


Dudley,  Rosenheim  and  Starling  (1927)  also  succeeded  in  the 
isolation  of  spermidine,  a  base  present  in  the  mother-liquor  after 
separation  of  spermine  phosphate;  spermidine  phosphate  is  much 
more  soluble  than  spermine  phosphate  and  crystallizes  from  the 
25%  ethanolic  mother-liquor,  after  the  removal  of  spermine  phos- 
phate, when  the  concentration  of  alcohol  is  increased  to  50%.  The 
properties  of  spermidine  are  similar  to  those  of  spermine.  It  gives 
the  same  pyrrole  reaction  and  behaves  in  an  identical  manner 
towards  precipitating  reagents  including  phosphotungstic  acid. 
Spermidine  is  optically  inactive,  and  yields  like  spermine,  the  semen- 
like odour  when  a  solution  of  its  chloroaurate  is  treated  with 
magnesium.  The  structural  formula  of  spermidine,  proved  by  syn- 
thesis, is  that  of  a-[y'-aminopropylamino]-(5-aminobutane: 


I  I 

NHa  NH2 


The  close  chemical  relationship  of  spermine  and  spermidine 
suggests  that  the  two  bases  may  be  related  metabolically.  Little, 
however,  is  as  yet  known  about  the  biogenesis  and  metabolism  of 
either  of  these  two  substances.  So  far  as  spermine  in  human  semen 
is  concerned,  there  can  be  little  doubt  that  its  high  concentration 
which  is  of  the  order  of  50-250  mg./lOO  ml.,  is  due  chiefly  to  the 
prostatic  secretion.  Fuerbringer  showed  in  1881  that  the  prostatic 
gland  contributes  by  far  the  greatest  part  of  seminal  spermine;  on 
addition  of  two  drops  of  1  %  solution  of  (NH4)2HP04  to  ten  drops 
of  freshly  collected  prostatic  fluid,  Fuerbringer  observed  an  almost 
instantaneous  formation  of  Boettcher's  crystals;  the  examination  of 
secretions  of  the  other  accessory  organs  gave  a  negative  result.  The 
concentration  of  spermine  in  the  human  prostate  is  subject  to  varia- 
tions but  exceeds  that  of  any  other  organ.  This  follows  both  from 
Harrison's  findings  (1931)  as  well  as  from  the  survey  carried  out  by 
Hamalainen  (1947)  who  determined  spermine  as  flavianate  after  pre- 
cipitation from  trichloroacetic  acid  extracts;  the  highest  values 
obtained  by  the  Finnish  investigator  in  the  different  organs  (ex- 
pressed as  mg.  spermine  phosphate  per  100  g.  tissue,  wet  weight) 

Spermine,  Choline,  Ergothioneine  167 

were,  prostate  456,  pancreas  77,  adrenal  58,  liver  43,  spleen  40, 
testis  29,  ovary  9.  It  is  doubtful  if  Fuerbringer's  (1886)  belief  in  the 
'vitalizing'  effect  of  the  prostatic  secretion  upon  spermatozoa  could 
be  applied  to  spermine  as  such;  Harrison  (1931,  1933)  was  unable  to 
detect  any  activating  influence  of  spermine  phosphate  on  human 
spermatozoa.  In  contrast  to  human,  bull  semen  contains  no  sper- 
mine; this  is  not  surprising  in  view  of  the  absence  of  a  true  functional 
prostate  in  the  latter  species. 

Oxidation  of  spermine  and  spermidine  by  diamine  oxidase 

Spermine  and  spermidine,  both  undergo  oxidation  in  the  presence 
of  diamine  oxidase,  an  enzyme  of  which  there  is  about  a  hundred 
times  more  in  human  seminal  plasma  than  in  blood  serum  (Zeller, 
1941);  this  finding  together  with  observations  by  earlier  investigators 
who  found  that  the  oxygen  uptake  of  human  semen  is  linked  with 
the  seminal  plasma  rather  than  the  spermatozoa,  led  Zeller  and 
Joel  (1941)  to  suggest  that  the  oxygen  consumption  in  human  semen 
is  mediated  chiefly  by  the  spermine-diamine  oxidase  system.  This 
requires  further  experimental  proof. 

State  of  spermine  in  semen 

An  interesting  but  as  yet  unsolved  problem  relates  to  the  state 
of  spermine  in  freshly  voided  semen.  Some  investigators  envisaged 
the  possibility  that  spermine  occurs  already  in  fresh  semen  as  a 
phosphate  salt  which  being  poorly  soluble,  separates  from  the  semen 
in  the  characteristic  shape  of  Boettcher's  crystals.  However,  in 
freshly  ejaculated  human  semen  there  is  not  enough  inorganic 
phosphate  to  combine  with  all  the  spermine  and  the  content  of 
inorganic  phosphate  increases  on  standing  owing  to  the  breakdown 
of  phosphorylcholine  (see  p.  170).  Furthermore,  it  has  been  the 
experience  of  all  those  who  tried  to  obtain  crystalline  spermine 
phosphate  from  semen,  that  a  successful  crystallization  can  best  be 
achieved  with  semen  which  has  been  allowed  to  stand  for  at  least  a 
few  hours  after  ejaculation  or  by  following  Fuerbringer's  recom- 
mendation and  treating  it  with  additional  phosphate.  Fuerbringer's 
(1881)  interpretation  of  his  own  findings  was  that  the  basic  com- 
ponent of  Boettcher's  crystals  in  ejaculated  semen  originates  in  the 

1 68  The  Biochemistry  of  Semen 

prostatic  secretion,  whereas  the  phosphoric  acid  is  derived  from 
some  other  source.  Recent  advances  in  this  field  favour  this  hypo- 
thesis and  indicate  that  the  formation  of  spermine  phosphate  takes 
place  only  after  the  ejaculation,  as  the  outcome  of  a  reaction  be- 
tween spermine  which  is  contributed  by  the  prostatic  secretion,  and 
phosphoric  acid,  which  accumulates  gradually  through  the  action 
of  the  seminal  phosphatases  upon  phosphorylcholine  and  perhaps 
also  upon  some  other  organic  phosphorus  compounds. 


The  Florence  reaction  in  semen 

Florence,  working  in  the  laboratory  of  forensic  medicine  in  Lyons, 
made  the  following  observation  in  1895;  if  material  stained  with 
]  luman  semen  is  extracted  with  water,  and  a  drop  of  this  extract  is 
mixed  on  a  microscopic  slide  with  a  strong  solution  of  iodine  in 
potassium  iodide  (2-54  g.  I2,  1-65  g.  KI,  30  ml.  water),  the  micro- 
scopic field  is  quickly  filled  with  a  mass  of  brown  crystals  which 
]-esemble  closely  Teichmann's  crystals  of  haemin.  Florence's  treatise 
'Du  sperme  et  des  taches  de  sperme  en  medecine  legal'  (1895/96) 
created  much  interest  in  forensic  medicine  and  led  promptly  to  the 
lecognition  of  his  test  as  a  useful  means  for  the  identification  of 
seminal  stains.  At  first,  a  hypothetical  substrate  called  'virispermine' 
was  held  responsible  for  the  formation  of  'iodospermine'  in  the 
Florence  reaction,  but  later  on  other  substances  came  under  investi- 
gation, including  choline.  All  doubts  concerning  the  nature  of  the 
Florence's  reaction  product  were  finally  dispelled  when  Bocarius 
(1902)  succeeded  in  converting  'iodospermine'  preparations  ob- 
tained from  human  and  stallion  semen,  into  a  crystalline  platinum 
compound  which  contained  31-62%  Pt  and  was  identical  in  every 
way  with  pure  choline  platinum  chloride  (31-64%  Pt).  Stanek's 
work  in  Prague  (1905,  1906)  had  shown  that  the  iodine  compound 
formed  in  Florence's  reaction  was  a  water-insoluble  periodide  of  the 
composition  of  an  enneaiodide,  corresponding  to  the  formula 
QHuNOFIg.  The  method  developed  by  Stanek  for  the  quantitative 
determination  of  choline  depended  on  the  analysis  of  nitrogen 
(Kjeldahl)  in  the  periodide  precipitate;  the  more  recent  quantitative 

Spermine,  Choline,  Ergothioneine  169 

method  of  Roman  (1930)  is  based  on  the  same  principle  but  involves 
an  analysis  of  iodine  instead  of  nitrogen 


CH3I     CH3 


Compared  with  other  animal  tissues  and  body  fluids,  semen  ranks 
as  one  of  the  richest  sources  of  choline.  It  owes  its  high  choline 
content  to  the  seminal  plasma  and  not  to  spermatozoa  as  such. 
In  rat,  Fletcher,  Best  and  Solandt  (1935)  found  the  following  dis- 
tribution of  total  choline  (mg./lOO  g.):  seminal  fluid  514,  brain  325, 
liver  260,  pancreas  232,  stomach  152,  uterus  74,  fat  23,  blood  22. 
The  composition  is  similar  in  other  species,  including  man,  where 
values  exceeding  2000  mg./lOO  ml.  semen  have  been  observed.  This 
may  explain  a  statement  by  Marcille  (1931)  that  a  positive  Florence 
reaction  can  be  obtained  with  dried  human  semen  even  when  it  is 
diluted  with  1000  parts  of  water.  However,  there  is  no  general 
agreement  about  the  sensitivity  of  Florence's  reaction.  In  fact,  many 
investigators  have  criticized  the  reaction,  mainly  because  the  same 
specimen  of  semen  will  occasionally  give  a  negative  reaction  at  first, 
and  a  positive  result  later.  This  peculiar  behaviour  of  human  semen 
was  elucidated  by  Kahane  and  Levy  (1936,  1937)  who  discovered 
that  human  semen  examined  immediately  after  ejaculation  contains 
practically  no  free  choline,  but  that  choline  accumulates  in  semen 
gradually  on  standing,  as  illustrated  by  the  following  experiment: 
from  3-5  ml.  semen  mixed  with  20  ml.  water,  consecutive  2  ml. 
samples  were  withdrawn  and  deproteinized  by  boiling  with  9  ml. 
ethanol  for  2  minutes;  the  quantitatively  collected  filtrates  were 
evaporated,  the  residues  extracted  with  dry  ether  and  redissolved  in 
water;  choline  was  precipitated  from  the  aqueous  extracts  with  the 
Reinecke  reagent  and  determined  bromometrically.  Results,  which 

Table  23.  Choline  in  human  semen  (Kahane  and  Levy,  1937) 

Time  after 

ejaculation       2  min.    10  min.     1  hr.      6  hr.     22  hr.    48  hr.    120  hr. 
Choline  liberated 

(mg.  per  100 

ml.  semen)    70    860    1600   2120   2030   2500   530 

170  The  Biochemistry  of  Semen 

are  given  in  Table  23,  show  a  sharp  increase  in  the  choline  content 
of  human  semen  during  the  first  hour  of  incubation,  and  the  rela- 
tively slow  accumulation  during  the  next  47  hours;  the  terminal 
decline  is  probably  due  to  bacterial  contamination. 

Enzymic  liberation  of  choline  froin  precursors  in  semen 

Following  up  their  observation  that  choline  accumulates  in  semen 
only  after  the  ejaculation,  Kahane  and  Levy  demonstrated  the  pre- 
sence in  fresh  semen  of  a  'precurseur  de  la  choline'  which  yields  free 
choline  as  a  result  of  hydrolysis  which  takes  place  in  semen  on 
standing.  Apart  from  the  seminal  plasma  itself,  they  found  the 
choline  precursor  in  various  reproductive  organs,  including  the 
testis  of  bull,  boar,  ram,  stallion,  rabbit  and  guinea-pig,  the  seminal 
vesicle  of  stallion  and  guinea-pig,  and  the  epididymis  of  boar  and 
ram,  but  not  the  prostate  of  dog,  stallion  or  ram.  However,  the 
prostate,  particularly  that  of  dog,  was  found  to  be  rich  in  the  enzyme 
which  splits  off  choline  from  the  precursor.  In  a  series  of  studies, 
Kahane  and  Levy  (1938,  1945,  1949)  have  shown  that  the  precursor 
is  a  water-soluble  compound  ('choline  hydrosoluble  combinee') 
which  behaves  like  glycerylphosphorylcholine,  and  yields  on  incu- 
bation with  prostatic  extracts  a  mixture  of  free  choline  and  in- 
organic phosphate;  the  quantity,  however,  of  liberated  choline  was 
found  to  be  far  in  excess  of  the  simultaneously  appearing  inorganic 

Phosphorylcholine  and  glycerylphosphorylcholine 

The  nature  of  the  phosphorus  compounds  in  semen  which  yield 
choline  after  ejaculation,  was  investigated  by  Lundquist  (1946, 
1947(7,  b)  and  by  Diament,  Kahane  and  Levy  (1952). 

In  human  semen  deproteinized  freshly  with  trichloroacetic  acid, 
the  Danish  investigator  found  110  mg.  acid-soluble  P/100  ml, 
including  10  mg./lOO  ml.  of  inorganic  phosphate.  On  neutraliza- 
tion with  barium  hydroxide  and  precipitation  with  2  vol.  of  ethanol, 
he  recovered  60-70%  of  the  phosphorus  in  the  filtrate  and  from  this 
he  obtained  by  precipitation  with  mercuric  chloride  a  fraction 
containing  nitrogen  and  phosphorus  in  a  ratio  of  approximately  1:1. 
The  phosphorus  compound  thus  separated  was  found  to  be  very 

Spermine,  Choline,  Ergothioneine  171 

resistant  to  acid  hydrolysis  and  no  choline  was  set  free  from  it  after 
an  hour's  hydrolysis  with  N-H2SO4  (100^),  long  enough  for  glyceryl- 
phosphorylcholine,  to  release  all  its  choline  in  a  free  form.  On  the 
other  hand,  under  the  influence  of  the  prostatic  secretion  the  com- 
pound yielded  equivalent  amounts  of  choline  and  inorganic  phos- 
phate. All  these  facts  pointed  to  the  identity  of  the  compound  with 
phosphorylcholine,  a  substance  previously  isolated  from  beef  liver 
(Inukai  and  Nakahara,  1935).  Lundquist  sought  to  obtain  proof  by 
preparing  the  calcium  salt;  this  he  found  to  be  identical  with  the 
calcium  salt  of  pure  phosphorylcholine,  C5Hi304NPClCa-4H20, 
obtained  synthetically  by  the  method  of  Plimmer  and  Burch  (1937). 


HO— P— O   -CHoCHa-N^ 

I  "       /|\ 

O  ChJ      CHp 



The  distribution  of  phosphorylcholine  in  the  human  reproductive 
organs  has  not  been  investigated  in  detail,  but  Huggins  and  Johnson 
(1933)  have  good  evidence  that  the  bulk  of  the  phosphorus  present 
in  the  human  seminal  plasma  is  derived  from  the  vesicular  secretion. 
From  this  Lundquist  infers  that  phosphorylcholine  is  formed  in  the 
seminal  vesicles,  and  that  the  dephosphorylation  is  initiated  at 
ejaculation,  as  a  result  of  contact  between  the  prostatic  secretion 
which  contributes  the  'acid'  phosphatase,  and  the  vesicular  secretion 
which  provides  the  substrate;  the  optimum  pH  for  the  dephos- 
phorylation of  phosphorylcholine  by  the  prostatic  phosphatase 
measured  in  acetate  buffer  solutions  is  about  6-3  (Lundquist,  1947a, 
b).  It  is  of  some  interest  to  recall  here  the  claim  put  forward  by 
Kutscher  and  Sieg  (1950)  that  preparations  of  both  the  'acid'  and 
the  'alkaline'  phosphatases  contain  pyrophosphorylcholine  as  a 
characteristic  constituent.  However,  Roche  and  his  colleagues  (1952) 
were  unable  to  detect  any  cophosphatase  activity  in  pure,  synthe- 
tically prepared  pyrophosphorylcholine. 

The  possibility  that  compounds  other  than  phosphorylcholine 
may  act  as  precursors  of  free  choline  in  semen  was  indicated  already 
by  the  earlier  observation  of  Kahane  and  Levy  that  the  quantity  of 

1 72  The  Biochemistry  of  Semen 

choline  liberated  after  ejaculation  exceeds  considerably  the  simul- 
taneously formed  inorganic  phosphate.  Following  up  this  observa- 
tion, the  French  investigators  accomplished  in  1952  the  isolation  of 
a  second  natural  precursor  of  choline,  namely  glycerylphosphoryl- 
choline,  from  the  seminal  vesicle  secretion  of  rats;  the  isolation  and 
identification  was  performed  as  a  ferric  chloride  compound  (Dia- 
ment,  Kahane  and  Levy,  1952,  1953).  A  similar  result  was  obtained 
by  Lundquist  (1953)  from  his  studies  on  the  secretions  of  the  seminal 
vesicles  in  rat  and  guinea-pig  and  the  glandula  vesicularis  of  rabbit. 

H,C— OH 

HC— OH  O 

HgC— O— P— O— CH2  CH2N+ 

O  CH3I     CH3 



Physiological  function  of  free  and  bound  choline 

The  occurrence  of  choline,  phosphorylcholine  and  glycerylphos- 
phorylcholine in  semen  and  in  the  accessory  secretions  naturally 
raises  the  problem  of  their  physiological  function.  One  possibility 
which  merits  serious  attention,  is  that  these  compounds  may  be 
bound  up  specifically  with  the  metabolism  of  phospholipids  in 
either  the  male  accessory  organs  or  in  the  spermatozoa. 

The  general  importance  of  choline  in  the  lipid  metabolism  of 
animals  was  first  brought  to  light  in  1932  when  Best  and  his  co- 
workers demonstrated  that  the  appearance  of  the  'fatty  livers'  in 
rats  fed  a  choline-deficient,  high-fat  diet,  could  be  prevented  by 
dietary  supplements  of  choline.  Researches  which  followed  estab- 
lished two  principal  functions  of  choline,  the  lipotropic  activity  and 
the  stimulating  action  on  the  turnover  of  phospholipids.  In  1939,  du 
Vigneaud  and  his  co-workers  discovered  that  choline  is  an  important 
dietary  source  of  methyl  groups  for  the  living  animal,  and  this  led 
to  the  recognition  of  choline  as  a  participant  in  transmethylation 
processes.  These  three  fundamental  functions  probably  represent 
the  clue  to  the  understanding  of  the  manifold  symptoms  associ- 
ated with  choline  deficiency.  Among  the  various  manifestations  of 

Spermine,  Choline,  Ergothioneine  173 

choline  deficiency  those  concerned  with  reproduction  are  particu- 
larly striking;  choline  is  known,  for  example,  to  be  essential  for  egg 
production  in  the  chicken,  as  well  as  for  normal  lactation  and 
nutrition  in  rats. 

The  role  of  choline  in  transmethylations  is  linked  with  the 
presence  of  the  trimethyl  quaternary  nitrogen.  It  is  worth  noting,  how- 
ever, that  while  the  phenomenon  of  transmethylation  is  common 
to  a  whole  group  of  compounds  bearing  labile  methyl  groups, 
the  lipotropic  activity  is  restricted  to  choline  and  a  few  closely 
related  derivatives.  One  of  the  lipotropically  active  derivatives  is 
phosphorylcholine  (Welch  and  Welch,  1938),  and  there  is  some 
evidence  that  the  incorporation  of  choline  into  phospholipids  pro- 
ceeds via  phosphorylcholine  (Wittenberg  and  Kornberg,  1953). 

A  further  possibility  regarding  the  function  of  choline  in  semen 
comes  to  mind;  choline  and  its  derivatives  belong  to  a  group  of  sub- 
stances endowed  with  well-defined  pharmacological  properties,  and 
it  is  not  improbable  that  the  base  itself  or  one  of  its  compounds  may 
exert  some  pharmacodynamic  effects  either  on  the  spermatozoa  or, 
perhaps,  on  some  parts  of  the  male  or  female  reproductive  tract. 
When  assayed  by  Goldblatt  (1935^)  on  the  m.  rectus  abdominis  of 
the  frog,  1  ml.  human  seminal  plasma  exhibited  roughly  the  same 
activity  as  l^g.  acetylcholine.  There  is,  however,  no  chemical  evi- 
dence to  show  that  the  substance  in  seminal  plasma,  responsible 
for  this  activity  is  in  fact,  acetylcholine. 

Choline  esterase 

It  has  been  claimed  that  sperm  motility  is  somewhat  increased 
by  acetylcholine,  and  depressed  by  eserine,  but  this  effect  has  never 
been  analysed  quantitatively  and  requires  confirmation.  There  is, 
on  the  other  hand,  sufficient  evidence  to  show  that  semen  contains 
choline  esterase  as  a  normal  constituent.  In  human  semen,  the  con- 
centration of  choline  esterase  was  found  to  be  low.  Zeller  and  Joel 
(1941)  using  the  manometric  method,  and  employing  a  rather  high 
concentration  of  acetylcholine  as  substrate,  found  that  the  quantity 
of  acetic  acid  liberated  by  1  ml.  semen  in  1  hr.  is  equivalent  to  not 
more  than  70  [A.  CO2,  as  compared  with  3600  /td.  in  blood  serum 
and  38000  /tl.  in  brain;  moreover,  the  bulk  of  activity  was  derived 
from  the  seminal  plasma  and  not  from  the  spermatozoa.  Boar 

1 74  The  Biochemistry  of  Semen 

semen,  on  the  other  hand,  has  been  found  by  Sekine  (1951)  to  be 
highly  active,  the  activity  being  more  concentrated  in  the  sperma- 
tozoa than  in  the  seminal  plasma.  According  to  this  author,  boar 
spermatozoa,  both  epididymal  and  ejaculated,  possessed  choline 
esterase  activity  as  high  as  that  of  brain,  whereas  the  seminal  plasma 
was  only  one-third  as  active  as  human  blood  serum.  Boar  sperma- 
tozoa, although  highly  active  against  acetylcholine,  were  found  at 
the  same  time  to  be  completely  inactive  against  benzoylcholine 
which  suggests  that  their  choline  esterase  is  of  the  'true'  or  'specific' 
type.  Results  obtained  on  ram  semen  (Legge  and  Mann,  unpublished 
data)  lead  to  a  similar  conclusion;  ram  spermatozoa  exhibited  a  high 
activity  at  low  concentrations  of  acetylcholine  but  were  poorly 
active  at  high  substrate  concentrations,  and  hydrolysed  efficiently 
acetyl-i9-methylcholine  but  were  ineffective  against  benzoylcholine. 
A  study  was  also  made  at  the  same  time  of  the  distribution  of  the 
enzyme  between  the  sperm-heads  and  -tails,  using  ram  spermatozoa 
disintegrated  with  glass  beads  in  the  Mickle  mechanical  shaker  (see 
p.  87).  Choline  esterase  occurred  mainly  in  the  tail  fraction. 


Ergothioneine  was  first  discovered  by  Tanret  (1909)  who  isolated 
it  from  rye  ergot.  Two  years  later,  Barger  and  Ewins  (1911)  identi- 
fied the  new  substance  as  a  betaine  of  thiolhistidine  (^-2-thiolglyoxa- 
line-4(5)-propiobetaine).  The  final  confirmation  of  the  structure  was 
provided  by  Heath,  Lawson  and  Rimington  (1950,  1951)  who  suc- 
ceeded in  synthesizing  ergothioneine  from  2-thiolhistidine;  the  latter 
is  an  amino  acid  which  so  far  has  never  been  found  in  nature 
but  was  prepared  synthetically  by  Harington  and  his  co-workers 
(Ashley  and  Harington,  1930;  Harington  and  Overhoff,  1933),  and 
shown  by  Neuberger  and  Webster  (1946)  to  be  unable  to  replace 
histidine  as  a  growth-promoting  factor  in  animals. 

/         \CH 

HS— C 


N^  +i(CH3)3 


Spermine,  Choline,  Ergothioneine  175 

Ergot  from  which  ergothioneine  has  been  obtained  in  yields 
varying  from  65  to  260  mg./lOO  g.,  remained  the  only  natural  source 
of  this  base  until  Hunter  and  Eagles  (1925,  1927)  isolated  from  pig 
blood  a  crystalline  substance,  named  at  first  'sympectothion',  which 
gave  with  phosphotungstic  and  arsenophosphotungstic  acid  reagents 
the  same  blue  colour  as  uric  acid.  Quite  independently,  a  blood 
constituent  with  similar  properties,  named  'thiasine',  was  obtained 
by  Benedict,  Newton  and  Behre  (1926).  Somewhat  later,  both 
sympectothion  and  thiasine  were  shown  to  be  identical  with  ergo- 
thioneine (Newton,  Benedict  and  Dakin,  1926;  Eagles  and  Johnson, 
1927).  Blood  ergothioneine,  or  'thioneine'  as  it  is  sometimes  called, 
occurs  only  in  the  erythrocytes  and  is  not  found  in  the  plasma.  In 
human  blood  there  is  no  more  than  about  2  mg./lOO  ml.,  but  in  the 
pig  there  may  be  as  much  as  26  mg./lOO  ml.  ergothioneine  (Hunter, 
1951).  Of  the  existing  methods  for  the  determination  of  ergothioneine 
that  of  Hunter  (1928,  1949),  based  on  the  diazo  reaction,  is  the  most 
sensitive,  specific  and  accurate. 

Isolation  of  ergothioneine  from  the  boar  seminal  vesicle  secretion 

It  has  been  known  for  quite  a  while  that  protein-free  extracts 
from  semen  exhibit  a  marked  reducing  power  towards  iodine, 
silver  nitrate,  2  :  6-dichlorophenol-indophenol,  and  potassium  per- 
manganate in  the  cold,  and  that  this  property  is  due  to  substances 
secreted  in  the  seminal  vesicle  fluid.  It  has  been  mostly  taken  for 
granted  however,  that  the  reducing  power  of  semen  is  due  to  ascorbic 
acid,  particularly  in  the  case  of  bovine  and  human  semen  (see  p.  23) 
and  no  attempt  was  made  to  strengthen  this  assumption  by  a  chemi- 
cal identification.  In  1951,  Leone  and  Mann  undertook  to  purify 
the  reducing  substance  from  the  boar  seminal  vesicle  secretion, 
which  being  available  in  relatively  large  quantities,  appeared  to 
offer  a  convenient  source  of  starting  material.  It  was  noticed  in  the 
course  of  the  purification  procedure  that  the  reducing  power  went 
parallel  with  three  other  chemical  properties  of  the  boar  vesicular 
secretion,  (i)  ability  to  reduce  phosphotungstic  acid  to  a  blue 
reaction  product,  (ii)  a  strongly  positive  diazo  reaction,  and  (iii) 
the  occurrence  of  organically-bound  sulphur  which,  however,  unlike 
that  present  in  glutathione,  cysteine  or  methionine,  could  be  oxidized 
and  readily  split  off"  as  inorganic  sulphate,  by  the  addition  of  mild 

176  The  Biochemistry  of  Semen 

oxidizing  agents  such  as  ferric  chloride  or  bromine  water.  These 
facts  suggested  that  the  reducing  substance  under  investigation  may 
be  the  imidazole  base  ergothioneine.  Further  purification  led  to  the 
isolation  from  1300  ml.  of  boar  vesicular  secretion  of  0-48  g.  crystal- 
line material  which  was  finally  identified  by  analysis  of  sulphur 
(140%),  nitrogen  (18-3%),  carbon  (471%)  and  hydrogen  (6-6%), 
and  by  other  chemical  means,  as  pure  ergothioneine,  C9H15N3O2S. 
With  the  isolation  of  ergothioneine  from  the  boar  vesicular  secre- 
tion and  boar  semen,  a  rather  unsuspected  and  abundant  source 
of  this  sulphur-containing  base  in  nature  has  been  discovered.  Un- 
like in  blood,  however,  ergothioneine  in  the  vesicular  secretion  is  an 
extracellular  constituent.  Moreover,  the  concentration  of  ergothio- 
neine in  this  accessory  secretion  is  much  higher  than  in  blood.  In 
samples  from  twenty  boars  of  the  Large  White  and  Essex  variety, 
we  found  from  29  to  256  mg./lOO  ml.;  the  average  was  79  mg./lOO 
ml.;  in  boar  semen  itself  the  concentration  is  about  15  to  20  mg./lOO 
ml.,  but  pig  urine  (boar  and  sow),  and  the  foetal  fluids  contain 
practically  no  ergothioneine  (Mann  and  Leone,  1953). 

The  function  of  seminal  ergothioneine  and  its  behaviour  towards 
sulphydryl-binding  substances 

If  ergothioneine  possesses  a  specific  physiological  role  in  boar 
semen,  this  may  well  be  linked,  through  its  reducing  sulphydryl 
groups,  with  a  protective  influence  on  spermatozoa.  Boar  semen, 
it  must  be  remembered,  differs  from  that  of  most  other  domestic 
animals  by  its  exceptionally  large  volume  and,  at  the  same  time, 
very  low  concentration  of  spermatozoa.  Moreover,  the  period  of 
time  required  for  the  completion  of  ejaculation  is  much  longer  in 
the  boar  than  in  other  animals.  Under  storage  conditions  in  vitro, 
the  survival  period  of  ejaculated  boar  spermatozoa  compares  on 
the  whole  unfavourably  with  that  of  ram  and  bull  sperm. 

The  results  of  investigations  by  Brachet  (1944)  and  MacLeod 
(1951)  have  brought  into  prominence  the  importance  of  reduced 
sulphydryl  groups  for  sperm  motility,  and,  as  previously  mentioned 
(p.  58),  substances  with  sulphydryl  groups  in  a  reduced  form,  such 
as  cysteine  or  reduced  glutathione,  protect  spermatozoa  in  vitro 
from  the  inhibitory  action  of  SH-binding  reagents.  It  is  probable 
that  glutathione  plays  actually  a  role  in  vivo  since  it  has  been  shown 

Spermine,  Choline,  Ergothioneine  111 

to  occur  normally  in  spermatozoa  (Infantellina,  1945;  Tesoriere 
and  Infantellina,  1946).  Our  researches  (Mann  and  Leone,  1953) 
demonstrated  that  ergothioneine,  which  is  a  natural  constituent  of 
the  seminal  plasma,  can  counteract  most  efficiently  the  sperm- 
paralysing  action  of  various  thiol-reagents,  including  not  only  the 
mercaptide-forming  and  alkylating  reagents  but  also  substances  such 
as  o-iodosobenzoate  which  act  by  oxidizing  compounds  with  SH- 
groups  to  the  corresponding  S-S  derivatives.  In  fact,  we  were  able 
to  demonstrate  the  mutually  antagonistic  action  of  ergothioneine 
and  o-iodosobenzoic  acid  in  experiments  with  boar  sperm  taken 

60  90 

Incubation  (min.) 
Fig.  16.  Effect  of  ergothioneine  on  boar  spermatozoa;  anaerobic  fructolysis 
at  37°  in  boar  epididymal  spermatozoa  to  which  fructose  was  added, 

2-5  mg.  fructose/10^  sperm;  O O,  no  additions;  x x, 

iodosobenzoate    (IO^^m);    • •,    iodosobenzoate   (10-^m)  + 

ergothioneine  (2  x  10" ^m). 

(Mann  &  Leone,  1953) 

178  The  Biochemistry  of  Semen 

directly  from  the  epididymis,  in  which  unlike  in  the  seminal  vesicle, 
ergothioneine  is  absent.  The  epididymal  spermatozoa  were  diluted 
with  Ringer-phosphate-fructose,  and  the  suspension  divided  in 
three  equal  portions;  in  one,  serving  as  a  control,  fructolysis  was 
measured  directly,  in  another  the  reaction  was  allowed  to  proceed 
in  the  presence  of  lO^^^.iodosobenzoate,  and  in  the  third  after  the 
addition  of  the  same  amount  of  inhibitor,  but  together  with  ergo- 
thioneine, the  latter  in  a  concentration  of  the  same  order  of  magni- 
tude as  actually  found  in  vivo  in  the  boar  vesicular  secretion.  It 
can  be  seen  from  Fig.  16,  that  whereas  the  presence  of  iodoso- 
benzoate  alone  checked  the  process  of  fructolysis,  the  inhibition  was 
prevented  by  the  simultaneous  addition  of  ergothioneine  so  that 
in  effect,  the  spermatozoa  were  able  to  proceed  with  the  normal 
utilization  of  fructose. 

Biogenesis  of  ergothioneine 

The  mechanism  of  ergothioneine  formation  in  the  boar  was 
studied  by  pursuing  the  fate  of  certain  orally  administered  com- 
pounds labelled  with  radioactive  sulphur,  253  (Heath,  Rimington, 
Glover,  Mann  and  Leone,  1953).  It  was  found  that  inorganic  sul- 
phate or  thiolhistidine  failed  to  provide  a  source  of  sulphur  for 
ergothioneine  in  the  boar;  in  this  respect,  the  behaviour  of  thiol- 
histidine is  of  particular  interest,  since  it  demonstrates  again  that 
physiologically  occurring  substances  need  not  necessarily  arise  from 
compounds  to  which  they  bear  a  close,  though  purely  structural, 
chemical  resemblance.  Methionine,  the  amino  acid  pivotal  in  bio- 
logical transmethylations,  was  capable  of  supplying  the  sulphur  for 
the  biosynthesis  of  seminal  ergothioneine.  The  spermatozoa  them- 
selves also  incorporated  sulphur  from  labelled  methionine  but  here, 
the  maximum  radioactivity  appeared  several  weeks  later  than  in 
the  seminal  plasma;  this  time-lag  is  presumably  due  to  the  fact  that 
the  processes  of  spermatogenesis,  sperm  maturation,  and  transport 
through  the  epididymis,  require  substantially  more  time  than  is 
needed  for  the  formation  and  secretion  of  seminal  plasma  in  the 
accessory  organs.  By  administering  to  a  living  animal  a  labelled 
compound  like  methionine  one  might  be  actually  able  to  deter- 
mine the  time  interval  required  for  the  processes  of  sperm  formation 
and  transport  in  the  male  reproductive  organs.  When  synthetic 

Creatine  and  Creatinine  179 

35S-labelled  ergothioneine  was  fed  to  the  boar,  some  of  it  was  ex- 
creted, unchanged,  in  the  semen.  This  provides  interesting  evidence 
of  the  passage  into  semen  of  a  substance  absorbed  from  the  ali- 
mentary tract. 

Since  ordinary  fodder  contains  no  ergothioneine,  there  remained 
the  possibiUty  of  its  microbial  formation  in  the  digestive  tract. 
This,  however,  was  not  borne  out  by  an  experiment  in  which 
aureomycin  was  fed  to  a  boar,  1  g.  daily  for  24  days,  but  did  not 
affect  in  any  way  the  level  of  ergothioneine  in  semen. 

Human  semen,  and  that  of  certain  other  mammals  so  far  inves- 
tigated, was  found  to  contain  only  a  trace  or  no  ergothioneine.  In 
the  bull,  ram,  and  in  man,  the  considerable  reducing  power  of  the 
seminal  plasma  towards  dichlorophenol-indophenol  is  derived  partly 
from  ascorbic  acid,  but  partly  also  from  other  reducing  substances 
which  await  proper  identification  (Mann  and  Leone,  1953).  An 
interesting  approach  in  this  direction  was  made  by  Larson  and 
SaHsbury  (1952,  1953)  who  reported  on  the  presence  in  bull  semen 
of  an  as  yet  unidentified  reducing  substance  characterized  by  a 
positive  reaction  with  sodium  nitroprusside,  and  of  sulphite. 


Occurrence  in  mammalian  semen,  and  in  the  sperm  and  gonads  of 

One  of  the  earliest  references  to  the  presence  of  creatine  and 
creatinine  in  male  reproductive  organs  is  to  be  found  in  a  paper  by 
Treskin  who  in  Hoppe-Seyler's  laboratory  in  1872,  isolated  016  g. 
pure  creatinine  from  two  pairs  of  bull  testes.  In  1923,  Steudel  and 
Suzuki  isolated  large  quantities  of  crystalline  creatinine,  together 
with  another  nitrogenous  base,  namely  agmatine,  from  ripe,  fresh 
testicles  of  herring.  Ilyasov  (1933),  using  the  colorimetric  method 

/NH.  /         \ 

HN=:C  COOH  HN=C  C=0 

I  "  I 

CH3  CH3 

Creatine  Creatinine 


1 80  The  Biochemistry  of  Semen 

based  on  Jaffe's  reaction,  determined  the  creatine  and  creatinine 
content  in  bull  and  stallion  semen.  The  mean  values  which  he 
reported  for  creatine  and  creatinine  were  (mg./lOO  ml.),  3  0  and  12-1 
in  the  bull,  and  6-2  and  3-7  in  the  stallion,  respectively.  In  the  boar, 
the  apparent  creatinine  content  has  been  stated  to  be  in  blood 
plasma  2-4,  in  whole  semen  0-3,  and  in  the  seminal  vesicle  secretion 
5-3  mg./lOO  ml.  (McKenzie  et  al.,  1938). 

The  generally  held  belief  that  in  invertebrate  animals  arginine 
occurs  in  place  of  creatine  is  not  supported  by  results  of  chemical 
analyses  of  gonads  and  sperm.  Greenwald  (1946)  found  in  the 
sperm-laden  nephridia  of  Echiurus  144  mg./lOO  g.  of  apparent  crea- 
tine, and  189  and  270  mg./lOO  g.  in  the  testes  of  Arbacia  and 
Strongylocentrotus,  respectively.  He  succeeded  in  preparing  sub- 
stantial quantities  of  pure  creatine  and  creatinine,  from  the  testes 
and  sperm  of  several  invertebrates,  including  the  sea-urchin  (Stron- 
gylocentrotus), Urechis  caupo,  Holothwia  tubiilosa,  and  Cuciimaria 
frondosa.  In  the  case  of  the  gonads  of  two  ascidia,  Microcosmus 
sulcatus  and  Boltenia,  which  contain  chromogenic  material,  no 
creatine  or  creatinine  could  be  isolated  but  019  g.  of  pure  betaine 
picrate  was  obtained  from  150  g.  of  mixed  gonads  of  Boltenia, 
indicating  a  concentration  of  at  least  44  mg.  of  betaine  per  100  g. 
of  tissue.  In  the  testes  of  arthropods,  molluscs,  and  of  a  nematode 
(Ascaris),  the  amount  of  chromogenic  material  was  so  low  as  to 
indicate  absence  of  creatine. 

Phosphocreatine  and  phosphoarginine 

The  possibility  that  spermatozoa  may  contain  creatine  in  the 
form  of  phosphocreatine  was  envisaged  by  Eggleton  and  Eggleton 
(1929)  who  found  that  the  testes  contain,  next  to  skeletal  muscle, 
the  second  largest  concentration  of  phosphagen.  Soon  after  the  dis- 
covery by  Parnas,  Ostern  and  Mann  (1934o,  b)  that  extracts  from 
skeletal  muscles  can  synthesize  phosphocreatine  from  creatine  and 
phosphopyruvic  acid,  the  same  enzymic  reaction  was  investigated 
in  bovine  epididymal  spermatozoa  by  Torres  (1935)  who  claimed 
that  bull  spermatozoa  are  definitely  capable  of  such  a  synthesis. 
Her  claim,  however,  has  been  refuted  by  Ivanov  (1937)  who  failed 
to  detect  any  synthesis  of  phosphocreatine  in  sperm,  although  he 
experienced  no  difficulty  in  confirming  our  results  on  muscles. 

Adrenaline  and  Noradrenaline  181 

More  recently,  Wajzer  and  Brochart  (1947)  reported  on  the  isola- 
tion from  boar  sperm  of  a  barium-precipitable  fraction  containing  a 
mixture  of  two  phosphagens,  phosphocreatine  and  phosphoarginine. 
The  distribution  of  the  two  phosphagens  in  the  gonads  and  in 
sperm  remains  open  to  further  investigations.  An  important  contri- 
bution in  this  field  was  made  by  Green wald  (1946)  who  isolated 
phosphocreatine  in  the  form  of  a  calcium  salt,  from  the  testes  of  the 


Occurrence  in  semen  and  accessory  organs 

Using  66%  ethanol  for  the  extraction  of  various  tissues  v.  Euler 
found  that  a  substance  closely  resembling  adrenaline  is  present  in  a 
particularly  high  concentration  in  the  prostate  gland  of  man,  dog, 
rabbit  and  guinea-pig,  in  the  seminal  vesicle  of  bull  and  ram,  and 
in  the  ampulla  ductus  deferentis  of  dog,  bull  and  ram;  the  amount 
of  the  active  substance  corresponded  to  1-5  //g.  of  adrenaline  per 
g.  of  fresh  tissue. 

Results  obtained  by  Brochart  (1948«)  with  the  colorimetric  method 
strengthened  the  view  that  adrenaline  occurs  as  a  normal  consti- 
tuent in  the  semen  of  bull  (1  /<g./ml.),  goat  (l-5-l-7/ig./ml.)  and  man 
(10-21  /ig./ml.);  but  later,  Beauvallet  and  Brochart  (1949)  came  to 
the  conclusion  that  in  the  bull  at  any  rate,  the  pressor  activity  of 
semen  is  due  partly  to  adrenaline,  and  partly  to  noradrenaline. 
H  H 

I  I 

HO— C  C CHaCH.,  HO— C  C CH^CHg 

II  I  II  II  I  I        I 

HO— C  C— H   OH  NH  CHg        HO— C  C— H   OH  NHg 

I  I 

H  H 

Adrenaline  (epinephrine)  Noradrenaline 

Enzymic  oxidation 

When  adrenaline  or  noradrenaline  are  added  to  bull  semen  in 
relatively  high  concentrations  (10-100  /^g./ml.),  the  aerobic  but 

182  The  Biochemistry  of  Semen 

not  the  anaerobic,  fructolysis  is  gradually  inhibited.  Brochart  (1951) 
attributed  this  effect  to  adrenochrome  and  noradrenochrome  which 
are  formed  aerobically  through  the  catalytic  action  of  the  cyto- 
chrome system  of  bull  spermatozoa  upon  adrenaline  and  noradren- 
aline, respectively.  Adrenochrome  itself,  added  to  bull  semen  in 
amounts  of  01-100  ^ag./ml,  produces  an  instantaneous  inhibition 
of  lactic  acid  formation  but  only  so  long  as  it  remains  in  the  oxidized 
form;  in  the  course  of  incubation  with  semen  it  becomes  gradually 
reduced  and  inactive. 

However,  an  alternative  mechanism  for  the  oxidation  of  seminal 
adrenaline  may  well  exist  since  Zeller  and  Joel  (1941)  have  found 
in  extracts  from  the  human  prostate  and  seminal  vesicle,  but  not  in 
the  seminal  plasma,  a  highly  active  monoamine  oxidase  (adrenaline 
oxidase).  The  reaction  catalysed  by  this  enzyme  follows  the  equation 

RCH^NR +O2+H2O  -^  RCHO+NHR'+HaOa 
and  leads  to  the  formation  of  hydrogen  peroxide  as  one  of  the 
reaction  products. 

Pharmacodynamic  properties 

An  interesting  synergistic  relationship  between  adrenaline  and 
seminal  plasma  was  reported  by  Goldblatt  (1935Z))  who  used 
isolated  seminal  vesicles  of  the  guinea-pig  as  his  test  object.  He 
observed  that  When  he  added  0-5-1  ml.  human  seminal  plasma  to  the 
medium  (30  ml.  oxygenated  Tyrode  solution)  there  was  usually 
no  response  from  the  vesicles;  if,  however,  a  small  amount  of 
adrenaline  was  added  first  and  a  considerable  interval  of  time 
allowed  to  elapse  until  the  only  activity  of  the  vesicle  was  an  occa- 
sional contraction,  then  the  addition  of  the  seminal  plasma  pro- 
voked a  succession  of  strong  contractions.  Adrenaline  in  doses  so 
small  as  to  be  entirely  devoid  of  activity  alone,  nevertheless  induced 
in  the  vesicles  a  condition  in  which  the  seminal  plasma  itself  or 
material  obtained  from  it  by  ethanolic  or  acetone  extraction,  were 
able  to  develop  to  the  full  their  pharmacological  activity.  This 
behaviour  of  adrenaline  led  Goldblatt  to  suggest  that  there  may  be 
a  sort  of  synergism  between  adrenaline  and  the  seminal  plasma. 
But  it  is  not  clear  as  yet,  whether  effects  of  this  kind  are  significantly 
related  to  the  function  of  either  the  male  or  the  female  reproductive 


Citric  Acid  and  Inositol 

Citric  acid.  Occurrence  and  distribution.  Influence  of  male  sex  hormone. 
Citric  acid  in  the  female  prostate.  Metabolism  and  role  of  seminal  citric 

Inositol.  Occurrence  and  distribution.  mesolnosiioX  as  a  major  con- 
stituent of  the  seminal  vesicle  secretion  in  the  boar.  Physiological  function. 
Relation  to  other  seminal  constituents. 

Citric  acid  and  inositol  which  will  be  considered  jointly  in  this 
chapter,  are  both  macro-constituents  of  the  seminal  plasma.  In  the 
past  these  two  chemical  substances,  much  like  fructose,  have  re- 
ceived attention  chiefly  from  plant  biochemists,  not  unnaturally, 
since  they  occur  in  plants  in  much  larger  quantities  and  more 
commonly  than  in  the  animal  kingdom.  Similarly,  ergothioneine  was 
at  first  associated  only  with  the  fungi,  until  at  a  much  later  date 
small  amounts  of  it  have  been  detected  in  red  blood  cells  and  more 
recently,  it  was  found  to  be  a  normal  constituent  of  boar  semen. 


Occurrence  and  distribution 

More  than  a  century  passed  after  Scheele's  (1784)  isolation  of 
crystalline  citric  acid  from  lemon  juice  before  this  tricarboxylic  acid 
was  discovered  in  the  animal  body  and  identified  as  a  major  chemical 
constituent  of  milk,  urine,  bone  and  semen.  The  discovery  in  semen 
was  made  in  Thunberg's  laboratory  at  Lund,  by  Schersten  (1929, 
1936),  who  noted  that  semen  rapidly  decolorizes  methylene  blue  on 
addition  of  'citrico-dehydrogenase',  an  enzyme  prepared  by  Thun- 
berg  from  cucumber  seeds.  This  observation  was  strengthened  by 
chemical  identification  based  on  isolation  of  crystalline  citric  acid 
and  the  preparation  of  pentabromoacetone,  a  derivative  formed 
from  citric  acid  on  oxidation  with  permanganate  and  bromine,  in 


184  The  Biochemistry  of  Semen 

a   reaction   described  in  1897  by   another   Swedisli  investigator, 





Citric  acid 

Schersten  enlarged  his  original  finding  by  noting  that  citric  acid 
in  semen  is  derived  from  the  male  accessory  organs  of  reproduction; 
in  man,  from  the  prostatic  secretion,  in  the  boar  and  bull,  from  the 
vesicular  secretion.  His  findings  have  since  been  confirmed  and 
extended  by  several  investigators.  In  nine  samples  of  human  pros- 
tatic secretion  Huggins  and  Neal  (1942)  recorded  values  ranging 
from  480  to  2688  mg.  citric  acid /1 00  ml.,  while  two  analyses  of 
human  seminal  vesicle  secretion  gave  15  and  22  mg./lOO  ml;  in  fif- 
teen specimens  of  human  semen,  the  values  ranged  from  140  to 
637  mg./lOO  ml.  A  survey  by  Harvey  (1951),  which  covered  725 
specimens  of  human  semen  from  371  donors,  revealed  contents 
ranging  from  0  to  2340  mg./lOO  ml.;  the  mean  value  of  citric  acid 
for  the  whole  group  was  479  mg./lOO  ml.  and  12-6  mg. /ejaculate. 
Citric  acid  also  occurs  normally  in  the  semen  of  other  mammalian 
species;  a  high  concentration  is  characteristic  for  the  bull  (510- 
1100  mg./lOO  ml.),  boar  (130  mg./lOO  ml.),  ram  (110-260  mg./lOO 
ml.),  and  rabbit  (110-550  mg./lOO  ml.);  rather  lower  concentrations 
are  found  in  stallions  (Humphrey  and  Mann,  1948,  1949).  In  some 
animals,  e.g.  the  bull,  ram,  boar,  and  stallion,  citric  acid  originates 
in  the  seminal  vesicle,  the  same  organ  which  also  secretes  fructose. 
In  other  species,  however,  the  two  substances  are  secreted  in  dif- 
ferent parts  of  the  male  reproductive  system  (Table  24).  In  the 
rabbit  citric  acid  is  limited  largely  to  the  gel-portion  of  semen,  and  it 
is  produced  by  the  glandula  vesicularis,  whereas  fructose,  it  will  be 
remembered,  is  secreted  also  in  the  prostate.  An  even  clearer  separa- 
tion occurs  in  the  rat  where  fructose  is  found  in  the  coagulating 
glands  and  in  the  dorso-lateral  prostate,  whereas  citric  acid  is 
produced  by  the  ventral  prostate  and  the  lateral  lobes  of  the  dorso- 
lateral prostate  (Fig.  4).  It  is  however,  probable  that  even  in  species 
such  as  the  bull,  where  citric  acid  and  fructose  are  found  side  by 

Citric  Acid  and  Inositol  185 

Table  24.  Distribution  of  citric  acid  in  male  reproductive  organs 
(Humphrey  and  Mann,  1949) 

Citric  acid 

Species                          Material 

(mg./lOO  g.  fresh  wt.) 

Boar:     Secretion  from  Cowper's 





Epididymal  semen 


Secretion  from  seminal 



Bull:      Testis 




Secretion  from  the  seminal 



Ampullar  semen 


Epididymal  semen 


Rabbit:  Epididymis 




Glandula  vesicularis 


Secretion  of  glandula 



Prostate  (I,  II  and  III) 


Cowper's  gland 




Rat:       Seminal  vesicle  proper 


Coagulating  gland 




Dorsolateral  prostate 


Ventral  prostate 


side  in  the  vesicular  secretion,  they  may  be  secreted  independently 
by  different  cells.  A  study  of  the  bull  seminal  vesicle  (Mann,  Davies 
and  Humphrey,  1949)  has  shown  that  the  secretory  epithelium  is 
composed  of  three  distinct  types  of  cells,  designated  A,  B  and  C, 
which  appear  to  be  concerned  in  the  secretory  processes,  but  react 
in  a  different  manner  to  several  histological  stains.  Type  B  con- 
sists of  lipid-laden  cells  mentioned  on  a  previous  occasion  (p.  133) 
whereas  A  and  C  cells  contain  no  lipid,  but  still  differ  materially 
from  each  other,  in  so  far  as  staining  is  concerned.  It  remains  for 
future  histochemical  studies  to  establish  their  specific  secretory 

186  The  Biochemistry  of  Semen 

Influence  of  male  sex  hormone 

There  is  a  close  relationship  between  the  formation  of  citric  acid 
in  the  male  accessory  organs  and  the  activity  of  the  testicular 
hormone  (Humphrey  and  Mann,  1948,  1949).  Following  castration, 
citric  acid  gradually  disappears  from  the  accessory  gland  secretions 
but  reappears  on  implantation  or  injection  of  testosterone.  In  this 
respect,  it  behaves  like  seminal  fructose,  except,  however,  that  in 
some  animals  (e.g.  rabbit)  the  postcastrate  disappearance  and  the 
hormone-induced  reappearance  of  citric  acid  in  the  seminal  plasma 
is  not  as  prompt  as  that  of  fructose.  The  'citric  acid  test'  which 
depends  on  the  relationship  between  the  formation  of  citric  acid  and 
androgenic  activity,  has  been  successfully  used  in  conjunction  with 
the  'fructose  test',  for  the  study  of  certain  endocrinological  prob- 
lems, such  as  the  time  relationship  between  spermatogenesis  and 
the  onset  of  secretory  function  in  male  accessory  organs  (Mann, 
Lutwak-Mann  and  Price,  1948;  Mann,  Davies  and  Humphrey, 
1949;  Mann,  1954);  formation  of  citric  acid  in  subcutaneous  trans- 
plants from  accessory  gland  tissues  (Lutwak-Mann,  Mann  and 
Price,  1949);  and  determination  of  androgenic  activity  in  ovarian 
hormones  (Price,  Mann  and  Lutwak-Mann,  1949,  1954). 

In  castrated  rats,  a  direct  relationship  exists  between  the  dose  of 
injected  testosterone  and  the  response  of  the  seminal  vesicle  to 
produce  citric  acid  (Mann  and  Parsons,  1950).  This  makes  it  pos- 
sible to  utilize  the  determination  of  citric  acid,  like  that  of  fruc- 
tose, as  a  sensitive  and  quantitative  assay  of  androgen  (Mann  and 
Parsons,  1950).  Removal  of  the  hypophysis  produces  the  same  end- 
result  as  castration  and  again,  the  secretion  of  citric  acid  by  the 
glandula  vesicularis  of  a  hypophysectomized  rabbit  can  be  restored, 
in  this  case,  either  by  testosterone  or  by  gonadotrophin  (Mann  and 
Parsons,  1950).  The  'citric  acid  test'  was  also  applied  in  studies 
concerned  with  the  influence  of  malnutrition  on  the  composition 
of  semen  (Lutwak-Mann  and  Mann,  1950^^,  b,  1951;  Mann  and 
Walton,  1953).  The  effect  of  malnutrition  manifests  itself  in  a  pro- 
gressive decline  of  the  citric  acid  level  in  semen  and  accessory  gland 
secretion  and  is  due  to  a  state  of  so-called  pseudo-hypophysectomy 
(see  p.  148). 

Citric  Acid  and  Inositol  187 

Citric  acid  in  the  female  prostate 

A  gland  corresponding  in  structure  to  the  male  prostate  gland 
develops  occasionally  in  the  female  body.  It  has  been  described  in 
women  but  most  studies  concerning  the  so-called  female  prostate 
have  been  done  with  rats  (cf.  Price,  1944;  Huggins,  1945;  Mann  and 
Lutwak-Mann,  1951^).  In  the  rat  this  organ  is  located  in  a  position 
similar  to  that  of  the  male  ventral  prostate  which  it  also  resembles 
histologically.  Ordinarily  the  incidence  of  prostate  gland  in  the 
female  rat  is  very  low  but  by  inbreeding  it  is  possible  to  increase  it 
to  80%  or  more.  With  rats  from  such  a  colony,  Price,  Mann  and 
Lutwak-Mann  (1949)  have  shown  that  the  analogy  between  the 
female  prostate  and  the  male  ventral  prostate  extends  to  the  chemical 
character  of  the  secretion  and  that,  like  its  male  counterpart,  the 
female  prostate  produces  citric  acid,  but  no  fructose.  Injections  of 
testosterone  brought  about  a  rapid  growth  of  the  gland,  and  a  sharp 
increase  in  the  output  of  citric  acid.  In  response  to  daily  adminis- 
tration of  200  /ig.  testosterone  propionate  continued  for  three  weeks, 
the  average  weight  of  the  female  prostate  rose  from  2  mg.  to  1 12  mg., 
and  the  average  content  of  citric  acid  in  the  gland  from  2  yt^g.  to 
125  /*g.;  in  male  rats  of  comparable  age  the  average  citric  acid 
content  of  the  ventral  prostate  was  121  /^g.  per  organ. 

Metabolism  and  role  of  seminal  citric  acid 

It  is  still  largely  a  matter  of  conjecture  how  citric  acid  is  formed 
in  the  accessory  organs.  The  rat  seminal  vesicle,  which  is  a  citric 
acid-producing  organ  is  at  the  same  time  remarkable  for  its  low 
content  of  aconitase  (Humphrey  and  Mann,  1949),  and  on  this 
ground  one  may  be  inclined  to  assume  that  perhaps  citric  acid 
accumulates  because  its  further  breakdown  is  prevented  by  the 
absence  of  this  enzyme.  On  the  other  hand,  however,  the  human 
prostate,  which  is  also  a  citric  acid-producing  organ,  has  been  found 
to  contain  aconitase  (Barron  and  Huggins,  \9A6b).  A  circumstance 
which  may  bear  some  relation  to  the  mechanism  of  citric  acid  accu- 
mulation in  the  bull  seminal  vesicle,  concerns  the  presence  in  this 
gland,  and  in  its  secretion,  of  a  heat-labile  factor  which  inhibits 
the  enzymic  breakdown  of  citrate  by  liver  tissue  (Humphrey  and 
Mann,  1949).  Yet  another  fact,  mentioned  briefly  in  conjunction 

188  The  Biochemistry  of  Semen 

with  the  general  chemical  properties  of  accessory  gland  secretions 
(p.  19)  concerns  the  high  transaminase  activity  in  the  human  pros- 
tate as  well  as  in  the  rat  ventral  prostate,  both  of  which  secrete 
citric  acid.  The  considerable  transaminase  activity  together  with  the 
occurrence  of  free  amino  acids,  including  glutamic  acid,  in  these 
two  glands,  point  to  the  possibility  of  citric  acid  being  formed  from 
oxaloacetic  acid  which  arises  from  glutamic  acid  as  a  result  of 
transamination  (Barron  and  Huggins,  1946o;  Awapara,  1952a,  b\ 
Awapara  and  Scale,  1952). 

As  to  its  physiological  role  in  semen,  the  available  evidence  does 
not  support  the  view  that  citric  acid  influences  markedly  the  aerobic 
or  anaerobic  metabolism  of  spermatozoa  (Humphrey  and  Mann, 
1949);  thus  the  beneficial  effect  of  citrate  on  sperm  motility  ob- 
served by  Lardy  and  Phillips  (1945)  may  be  due  to  a  cause  other 
than  direct  utilization  by  sperm.  It  is  conceivable  that  citric  acid 
is  connected  with  the  coagulation  and  liquefaction  of  semen  and 
with  the  calcium-binding  capacity  of  seminal  plasma.  In  this  con- 
nection one  may  recall  the  finding  of  Huggins  and  Neal  (1942) 
that  citrate  in  human  semen  causes  prolonged  coagulation  of  mix- 
tures of  blood  and  seminal  plasma,  and  that  this  delay  in  clotting 
can  be  effectively  counteracted  by  calcium  ions.  The  function  of 
citric  acid  as  a  binding  substance  for  calcium  has  been  envisaged 
both  by  Schersten  (1936)  and  Huggins  (1945)  and  it  is  certainly  sig- 
nificant that  milk  and  bones,  both  rich  in  citrate,  also  have  a  high 
calcium  content.  Perhaps  in  the  absence  of  citric  acid  in  the  prostatic 
secretion,  there  would  be  an  even  higher  incidence  of  calculi  and 
stones.  The  possibility  of  a  link  with  the  hyaluronidase  activity 
cannot  be  excluded,  as  indicated  by  Baumberger  and  Fried  (1948) 
who  found  that  citrate  exerts  a  protective  action  against  so-called 
antinvasin  in  vitro.  Lundquist  (1947Z?),  however,  believes  that  citrate 
may  act  as  an  activator  of  the  prostatic  'acid'  phosphatase.  Lastly, 
let  it  be  remembered  that  citric  acid,  in  combination  with  potassium 
and  sodium  ions,  may  play  a  part  in  maintaining  the  osmotic 
equilibrium  in  semen.  Our  own  studies  on  the  boar  vesicular  secre- 
tion (Mann,  1954)  point  in  this  direction. 

Citric  Acid  and  Inositol  189 


Occurrence  and  distribution 

Inositol  was  first  discovered  in  1850  by  Scherer,  at  Wiirzburg, 
who  isolated  it  from  the  mother-liquor  remaining  after  the  separa- 
tion of  creatine  from  beef  meat,  as  a  crystalline,  colourless  and  dis- 
tinctly sweet-tasting  substance.  He  named  it  'inosit'  to  underline  its 
origin  from  muscle,  and  showed  that  its  composition  and  properties, 
except  for  lack  of  reducing  capacity  are  similar  to  those  of  a  hexose. 
In  1887  Maquenne  proved  that  this  non-reducing  and  optically- 
inactive  compound  is  not  a  sugar  in  the  strict  sense  but  a  hexitol 
derived  from  cjc/ohexane.  In  the  years  which  followed,  Bouveault 
(1894)  and  others  brought  forward  evidence  for  the  existence  of 
several  cyclitols  derived  from  cjc/ohexane;  since  then  it  became 
customary  to  define  the  compound  from  muscle  as  m^^oinositol,  in 
distinction  to  the  other  isomers.  The  configuration  of  meso'mosiioX 
was  finally  established  in  1942,  by  Dangschat  in  Fischer's  laboratory, 
and  by  Theodore  Posternak,  in  Switzerland. 

H        H 

1         I 

C C 

H      / 1  I  \    OH 

1/    OH    0H\| 

c  c 

I  V     OH     H   /  I 

I  I 

H        OH 


Apart  from  muscle,  inositol  has  also  been  isolated  from  urine 
(Cloetta,  1856),  and  from  green  beans  (Vohl,  1856).  Later,  several 
plants  were  found  to  yield  on  extraction  such  very  large  amounts 
of  this  cyclitol,  that  like  citric  acid,  me^oinositol  came  to  be 
regarded  generally  as  a  typical  plant  constituent,  a  view  strengthened 
by  the  discovery  of  phytic  acid  (inositol  hexaphosphate)  in  grain, 
and  lipositol  (a  monophosphoinositol-containing  phosphatide)  in 
soya-bean.  The  interesting  history  of  these  and  later  developments 
in  the  biochemistry  of  inositol  will  be  found  in  the  monograph  by 

190  The  Biochemistry  of  Semen 

Fleury  and  Balatre  (1947)  and  in  the  reports  by  Courtois,  Fleury, 
Posternak,  and  Schopfer,  forming  part  of  a  (1951)  symposium. 

Inositol  as  a  major  constituent  of  the  seminal  vesicle  secretion  in  the 

In  1951,  in  the  course  of  investigations  on  ergothioneine,  the 
author  noticed  that  a  large  ethanol-precipitable  fraction  could  be 
separated  from  the  boar  vesicular  secretion,  containing  no  ergo- 
thioneine, citric  acid,  or  fructose,  and  almost  free  from  sulphur, 
nitrogen,  or  phosphorus.  On  further  purification,  from  1  litre  of 
the  vesicular  secretion,  18  g.  of  crystalline  material  was  obtained 
which  had  a  pronounced  sweet  taste,  but  was  non-reducing  and 
optically  inactive.  The  substance  had  a  m.p.  of  225°  which  is  that 
of  pure  m^'^cinositol,  and  contained  40-28 °o  carbon  and  6-79% 
hydrogen,  as  against  40-ll°o  carbon  and  6-66%  hydrogen,  theoreti- 
cally expected  from  inositol.  The  Scherer-Salkowski  reaction  per- 
formed with  01  mg.  was  strongly  positive,  and  on  oxidation  with 
periodic  acid  the  substance  isolated  from  the  seminal  vesicle  secretion 
showed  a  titration  curve  identical  with  that  of  pure  mesomo?,\Xo\. 

This  and  subsequent  experiments  (Mann,  1951c,  1954)  showed 
that  the  boar  vesicular  secretion  is  the  richest  source  of  free 
inositol  in  nature,  and  that  between  40  and  70%  of  the  dialysable 
contents  of  this  biological  fluid  is  made  up  of  inositol.  Table  25 
shows  the  results  of  chemical  analyses  of  the  vesicular  secretion 
from  five  boars,  carried  out  in  each  instance  on  fluids  collected 
separately  from  the  left  and  right  gland.  As  can  be  seen,  the 
inositol  content  of  these  fluids  was  208  to  2-64%,  the  variations 
being  much  smaller  than  they  would  be  in  the  case  of  fructose  or 
citric  acid.  It  is  also  of  some  interest  to  note  that  the  left  and  the 
right  seminal  vesicle  produced  secretory  fluids  which  were  alike  in 
quantitative  composition.  The  average  values  (mg./lOO  ml.)  based 
on  the  analyses  of  five  pairs  of  secretions  were:  fructose  65,  citric 
acid  381,  ergothioneine  91,  inositol  2414. 

Inositol  is  restricted  in  its  distribution  to  the  seminal  vesicle,  and 
is  not  present,  at  any  rate  in  appreciable  quantities,  in  the  secretions 
from  the  boar  epididymis  or  Cowper's  gland.  Furthermore,  it 
appears  to  be  rather  specific  for  the  boar,  so  far  as  can  be  judged 
from  preliminary  experiments  carried  out  with  the  semen  of  other 

Citric  A cid  and  Inositol  191 

Table  25.  Individual  variations  in  the  composition  of  boar  vesicular 
secretion  (Mann,  1954) 

(Analyses  carried  out  separately  on  the  fluids  collected  from  the  left 
(L)  and  right  (R)  seminal  vesicle  of  five  boars,  nos.  I-V.) 

I  II  III  V  V 

Total  volume  of  secretion 

from  both  vesicles  (ml.) 






Total  weight  of  the  empty 

vesicles  (g.) 






Concentration  in  the  vesicu- 

lar secretion 

(mg./lOO  ml.) 














Citric  acid 














=  (L) 

































(R)     2355    2610    2640    2345    2080 

Species.  Bull  semen  is  poor  in  inositol,  and  human  semen,  according 
to  Nixon  (1952),  contains  usually  less  than  01%. 

Physiological  function 

In  the  past,  the  physiological  function  of  inositol  has  been 
associated  mainly  with  nutrition,  particularly  since  Woolley's  (1944) 
important  observations  on  the  curative  effect  of  inositol  in  a 
dietary  dystrophy  in  mice,  coupled  with  retarded  growth  and 
alopecia.  Inositol  has  also  been  known  to  remedy  a  certain  type 
of  fatty  liver  in  rats  (Gavin  and  McHenry,  1941;  MacFarland  and 
McHenry,  1948),  and  its  role  as  a  lipotropic  factor  has  been  stressed 
repeatedly.  An  observation,  however,  which  merits  particular  atten- 
tion in  view  of  its  bearing  on  animal  reproduction,  concerns  a 
peculiar  disturbance  in  hamsters:  if  hamsters  are  raised  on  an 
inositol-deficient  diet,  they  tend  to  produce  dead  litters  or  die  in 
parturition  (Hamilton  and  Hogan,  1944).  Yet  another  aspect  of  the 

192  The  Biochemistry  of  Semen 

function  of  inositol  became  apparent  when  Chargaff  and  his  co- 
workers (1948)  reported  that  in  tissue  cultures  inositol  protects 
dividing  fibroblasts  from  the  toxic  effect  of  colchicine  and  other 
mitotic  poisons. 

It  remains  for  future  studies  to  determine  more  fully  the  function 
of  inositol  in  boar  semen.  Judging  from  the  author's  own  experi- 
ments there  is  little  evidence  that  inositol  is  metabolized  directly  by 
spermatozoa.  One  is  inclined  to  assume  for  inositol  a  role  in  the 
maintenance  of  the  osmotic  equilibrium  in  boar  seminal  plasma, 
seeing  that  the  seminal  vesicle  secretion,  unlike  other  body  fluids 
of  the  pig,  is  almost  completely  devoid  of  sodium  chloride  (Mann, 
1953,  1954). 

Relation  to  other  seminal  constituents 

The  mechanism  by  which  inositol  is  formed  in  mammals  is 
obscure  but  two  tentative  hypotheses  have  been  put  forward  in  the 
past.  One  involves  its  formation  from  a  derivative  of  phosphoinositol 
such  as  for  instance,  lipositol.  Fischer  (1945)  however,  believes  that 
inositol  acts  in  the  animal  body  as  a  sort  of  chemical  intermediary 
between  the  sugars  and  certain  aromatic  substances,  or  alternatively, 
as  a  reserve  carbohydrate  for  hexoses.  The  close  structural  similarity 
of  inositol  to  glucose  as  well  as  to  fructose,  is  certainly  a  point 
which  must  be  considered  in  future  investigations  on  the  origin 
of  seminal  inositol. 


A  STRIKING  feature  of  semen  which  did  not  escape  Leeuwenhoek, 
and  which  has  been  abundantly  and  repeatedly  confirmed  since,  is 
the  extraordinary  diversity  of  shape  and  structure,  encountered 
among  the  spermatozoa  of  different  species.  It  even  led  Wagner  and 
Leuckart  (1852)  to  state  'that  one  may  often  safely  venture  to  infer 
from  the  specific  shape  of  these  elements  the  systematic  position 
and  the  name  of  the  animals  investigated'.  Similarly,  anatomists  and 
physiologists  aUke,  have  long  accepted  as  natural  the  existence  of 
remarkable  species  variations  in  the  form  and  size  of  the  male 
accessory  glands,  the  organs  responsible  for  the  elaboration  of  that 
apparently  indispensable  adjunct  of  spermatozoa,  the  seminal 
plasma.  It  behoves  us,  I  feel,  to  adopt  a  similarly  enlightened  atti- 
tude of  mind  towards  the  chemistry  of  semen.  Is  it  not  rather  un- 
reasonable to  expect  that  chemical  findings  made  with  the  semen  of 
one  species  must  needs  extend  to  that  of  others?  The  fact  that  a 
given  substance  is  found  in  substantial  amounts  in  the  semen  of  one 
species,  but  is  missing  in  others,  by  no  means  detracts  from  its 
physiological  value:  on  the  contrary,  it  is  highly  probable  that  such 
species-restricted  occurrence  is  intimately  linked  with  some  other 
biological  characteristics,  peculiar  to  certain,  but  not  necessarily 
all,  animal  species. 

A  critical  approach,  free  from  bias,  is  also  called  for  in  the 
comparative  evaluation  of  the  morphological  and  chemical  findings 
in  semen.  To  expect,  as  has  been  done,  the  existence  of  a  strict  cor- 
relation between  say,  the  fructose  level  in  seminal  plasma  and  sperm 
density  in  semen,  is  no  more  justifiable  than  to  look  for  a  relation- 
ship between,  for  instance,  the  glucose  level  in  blood  plasma  and 
the  number  of  red  cells  in  blood.  Similarly,  although  the  secretion 
of  fructose  in  the  accessory  organs  depends  closely  upon  the  activity 
of  the  male  sex  hormone,  it  would  be  mistaken  to  attribute  the  level 
of  fructose  in  semen  to  the  influence  of  this  hormone  alone,  because 
in  reality  it  is  conditioned  by  a  multitude  of  other  factors,  including 
the  general  nutritional  state  of  the  body,  size  and  storage  capacity 


194  The  Biochemistry  of  Semen 

of  the  accessory  glands,  frequency  of  ejaculation,  volume  of  semen» 
ratio  between  sperm  and  seminal  plasma,  and  last  but  not  least,  the 
blood  glucose  level.  Above  all,  it  is  essential  to  bear  in  mind  that 
profound  changes  in  the  composition  of  semen,  elicited  in  response 
to  drastic  experimental  procedures  like  castration  or  hypophy- 
sectomy,  are  unlikely  to  be  equalled  in  extent  by  those  encountered 
in  hormonally  deficient  humans  or  in  large  domestic  animals. 

Having  thus  come  to  the  end  of  my  discourse,  I  would  like  to 
leave  the  last  word  with  Leeuwenhoek;  when  reporting  in  1677  to  the 
Rt.  Hon.  the  Viscount  Brouncker,  President  of  the  Royal  Society, 
upon  the  progress  of  his  researches  on  semen,  he  felt  it  incum- 
bent upon  him  to  add:  .'If  your  Lordship  should  consider  that  these 
observations  may  disgust  or  scandalize  the  learned,  I  earnestly  beg 
your  Lordship  to  regard  them  as  private  and  publish  or  destroy 
them,  as  your  Lordship  thinks  fit.' 


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Abalone,  egg-membrane  lysin,  95 
Abnormalities  in  semen,  11,  39 
Absorption     spectrum     of    cyto- 
chrome,   90;    intensification    by 
liquid  air,  65,  91 
Accessory  glands,  structure,  15-16; 
secretory  function,  15-29,  137- 
50,     167,     172,     176-7,     184-8, 
191-4;  onset  of  activity,  140-1, 
186;    action    of    secretions    on 
sperm,   24,   55;   dependence  on 
androgens,    67,    139^6,    186-7; 
pharmacological    effects,    25-7; 
after  irradiation,   59-60;  lipids, 
130-3;  utilization  of  sugars,  150; 
fructose,  137-8;  citric  acid,  184-8; 
changes  in  malnutrition,  148,  186 
Acetal  phospholipids,  126-7 
Acetic  acid,  formation,  51;  as  sub- 
strate, 52 
Acetylcholine,  27,  173 
Acidity,  effect  on  sperm,  60-1 
Acid  phosphatase,   in  semen  and 
accessory  secretions,  16-17, 118- 
20;  during  spermatogenesis,  5;  in 
galea  capitis,   13;  histochemical 
detection,    13;    dependence    on 
androgens,  68,  118-20;  purifica- 
tion,   119-20;    role    in    choline 
formation,     119,    168;    role    in 
spermine  phosphate  formation, 
167-8;  activation  by  citric  acid, 
Acid-soluble     phosphorus,     20-2, 

32-3,  36,  107,  128,  191 
Aconitase,  187 
Acrosome,    12;   PAS-reaction,    12; 

indophenol  reaction,  90 
Adenine,  100,  101,  116,  155 

Adenosine  diphosphate,  156 
Adenosinetriphosphatases,  86, 122- 

3;  and  sperm  activity,  14,  155-8 
Adenosine  triphosphate,   distribu- 
tion and  function  in  semen,  107, 

129,  155-8;  after  irradiation,  59; 

during  senescence,  77 
Adenylic  acid,  dephosphorylation, 

Adrenaline  in  semen  and  accessory 

organs,  27,  181-2 
Adrenaline  oxidase,  182 
Adrenochrome,   effect   on   sperm, 

Age  of  sperm  {see  also  ripening), 

Agglutination  of  sperm,  70,  79,  114 
Agmatine,  179 
Alanine,  in  semen  and  reproductive 

organs,  86,  106,  113;  in  diluents, 

Albumin,  in  semen,  112;  effect  on 

sperm,  76 
Albumose  in  semen,  1 1 1 
Algae,  carotenoids  in  reproduction, 

Alkaline  phosphatase,  120-1,  150; 

during  spermatogenesis,  5;  role 

in  fructose  formation,  150-1 
Alkalinity,  effect  on  sperm,  61 
Alkaloids,  effect  on  sperm,  56;  in 

chemotaxis,  71-2 
AUelostasis,  74 
AUomyces,  carotenoids  in  gametes, 

Alloxan  diabetes,  effect  on  seminal 

fructose,  146 
Amino  acid  oxidase  in  sperm,  117 
Amino  acids,  free  in  semen  and 

prostate,  19-20,  86,  111-13,  188; 

in  seminal  proteins,  84-7, 106-10; 



The  Biochemistry  of  Semen 

Amino  acids  (contd.) — 

in  diluents,  76,  114;  effect  on 
sperm,  76,  114;  after  castration, 

Aminopeptidase,  1 1 6 

Aminosugar,  21,  36,  112 

Ammonia,  21,  32-3;  formation, 
111,  116-17 

Amphibia  {see  also  frog),  sperm- 
affecting  agents,  54,  95;  nucleic 
acid,  105 

Ampulla,  secretory  function,  15, 
24,  113,  138,  181,  185 

Androgamones,  70-1;  95-6 

Androgens,  in  semen,  67-9;  in 
ovaries,  144;  indicator  tests  based 
on  semen  analysis,  67-8,  118-19, 
139-45,  186-7;  effect  on  acces- 
sory organs,  67-8 

Antibiotics,  use  in  semen  diluents, 

Antibodies  in  semen,  79-80 

Anti-fertility  factor,  98 

Antifertilizin,  70 

Antifibrinolysin,  115 

Antigens  in  semen,  79-80 

Arbacia  {see  also  sea-urchin),  pH 
of  semen,  44;  sperm  nucleic  acid, 
104;  creatine,  180;  gelatin-liquefy- 
ing enzyme  and  mucopoly- 
saccharase,  96;  echinochrome, 
70;  protein,  84;  effect  of  seminal 
plasma  and  amino  acids  on 
sperm,  76 

Arbacine,  108 

Archegonia,  chemotropic  sub- 
stances, 72 

Arginine,  free,  113;  protein-bound, 
84,  106-8 

Arniadillidium  vulgare,  sperm  sur- 
vival in  the  female  tract,  9 

Arsenicals,    effect      on      sperm, 

Artificial  diluents,  55,  73,  76,  79-82 

Artificial  insemination,  3,  38-40, 

Arvelius  albopunctatus,   PAS-reac- 

tion  in  the  acrosome,  11-12 
Ascaridine    in    Ascaris    megaloce- 

phala,  85 
Ascidia,  betaine  in  gonads,  1 80 
Ascorbic  acid,  20,  23,  32-3,  175, 

Asellus  aquaticus,  spermatogenesis 

and  ribonucleic  acid,  5 
Ash  content  of  semen,  32-3,  43,  87 
Aspartic   acid,    free,    86,    113;    in 

proteins,  85 
Ass  semen,  2,  43 
Aureomycin,  in  diluents,  81;  effect 

on  biosynthesis  of  ergothioneine, 

Axial  filament,  13 
Azide,  effect  on  sperm,  56,  91 
Azoospermia,  due  to  hyperpyrexia, 

62;  urinary  excretion  of  steroids, 

62;  fructose  and  fructolysis,  46, 


Bacteria  in  semen,  39,  81 

Bacteriostatic  properties  of  sper- 
mine, 162 

Barberio  reaction,  163^ 

Bat  semen,  volume,  2;  sperm  dens- 
ity, 2,  82;  survival  of  spermato- 
zoa, 6,  9,  82;  clotting,  28; 
dilution  effect,  82 

Bee,  clotting  of  semen,  28 

a-Benzoinoxime  (cupron),  as  a 
metal-binding  agent,  76 

Benzoquinone,  effect  on  sperm,  57 

Betaine,  in  gonads  of  ascidia,  180 

Bicarbonate,  in  semen  and  acces- 
sory secretions,  18,  22,  32-3;  in 
diluents,  81 

Blood  glucose,  as  precursor  of  fruc- 
tose, 149 

Blood  plasma  and  serum,  effect  on 
sperm,  55,  79 

Boar  accessory  organs,  16,  35-6 

Boar  semen,  volume  and  density,  2, 
35-6;   dry  weight,   32;  physico- 



chemical  properties,  41-4,  60; 
sperm  structure,  1 1 ;  gelation,  28, 
35;  protracted  period  of  ejacula- 
tion, and  fractions,  28,  35-7; 
nitrogenous  compounds,  32;  cit- 
ric acid,  36,  184-8;  creatinine, 
phosphocreatine  and  phospho- 
arginine,  180-1;  ergothioneine, 
36-7,  175-9;  fructose,  32,  36-7, 
136;  lactic  acid,  36;  lipids,  32, 
126;  mineral  constituents,  32,  36; 
protein  and  proteose,  32,  113; 
choline  esterase,  173-4;  hyalu- 
ronidase,  94;  respiration,  50,  60; 
resistance  to  dilution,  78;  time 
required  for  spermatogenesis  and 
ripening,  178-9 

Boar  vesicular  secretion,  contribu- 
tion to  semen,  35-7;  composi- 
tion, 20-4,  35-7,  175-6,  185,  191; 
individual  variations,  191;  fruc- 
tose, 35-6,  191;  inositol,  190-2; 
ergothioneine,  175-6;  citric  acid, 
184-8;  creatinine,  180;  osmotic 
equilibrium,  188,  192 

Boettcher  crystals,  161 

Bombyx  mori,  sperm  and  accessory 
secretions,  25 

Bouchon  vaginal,  28-9 

Bracken  sperm,  72,  92 

Broad  helix,  see  mitochondrial 

Buffering  capacity  of  semen,  40,  45 

Bull  ampullar  secretion,  adrena- 
line, 181;  citric  acid,  185;  fruc- 
tose, 16 

Bull  semen,  volume  and  density,  2; 
dry  weight,  32,  85;  ash,  87; 
physico-chemical  properties,  7, 
8,  41-5,  60;  nucleic  acid,  5,  105; 
metabolism  and  its  relation  to 
motility  and  fertihty,  45-53,  58, 
77,  128,  152-4;  seminal  fractions, 
38;  time  required  for  sperm  to 
reach  the  ovum,  9;  sperm  struc- 
ture, 10-15;  separation  of  sperm 

parts,  85-7;  PAS-reaction,  12; 
spermatogenesis  and  vitamin  A, 
72;  malnutrition  effects,  149;  cas- 
tration and  hormone  effects, 
142-9;  vasectomy  effect,  113, 138; 
androgen  content,  68-9;  diluents, 
77-8,  80-2;  composition,  32-5, 
84-7;  proteins  and  proteose,  32, 
84-7,  110;  sulphur,  85-7,  179; 
amino  acids,  85-7,  113;  amino 
acid  oxidase,  117;  phosphorus 
compounds,  32,  125-8,  155,  180; 
phosphatases,  120-3;  hyaluroni- 
dase,  94;  iron,  88,  109;  haematin, 
88;  catalase,  92;  cytochrome,  90; 
nitrogenous  bases,  180-2;  uric 
acid,  32,  123;  citric  acid,  32-3, 
184-8;  inositol,  191;  lipids,  125- 
8;  reducing  substances,  23-4,  179; 
fructose  and  fructolysis,  32-5, 
46-8,  135-9,  152-4 
Bull  seminal  vesicles,  secretory 
epithelium,  185;  secretory  func- 
tion 16,  20,  34,  38,  137;  hormonal 
aspects,  137-42;  role  in  fructose 
formation,  16, 137-8, 142, 149-51 
Bull  vesicular  secretion,  composi- 
tion, 20^;  lipid,  133, 185;  adrena- 
line, 181;  fructose,  138,  142;  citric 
acid,  184-8;  flavin,  22;  reducing 
substances,  23-4;  phosphatases, 
118-23;  xanthine  oxidase,  123 
Bursa  copulatrix,  role  in  insects,  9 
Butylquinone,  effect  on  sperm,  57 
Butyric  acid,  as  substrate,  52 

Calcium,  18,  21,  32-3,  36,  77,  132, 

Cap  (sperm-cap),  12-13 

Capacitation,  8-9 

Carbohydrate  {see  also  individual 
sugars),  in  gynogamone,  71;  in 
semen,  21-4,  30-8,  135-7;  meta- 
bolism, 45-53,  134-59 

Carbon  dioxide,  see  bicarbonate 

Carbonic  anhydrase,  19,  87,  89 


The  Biochemistry  of  Semen 

Carbon  monoxide,  effect  on  sperm, 
73,  91-2;  reaction  with  cyto- 
chrome oxidase,  91 

Carotenoids,  associated  with 
gametes  and  reproduction,  72-3 

Carp  sperm,  104,  106,  181 

Castration  effects,  113,  119,  139- 
45,  186 

Cat,  21;  lipid  bodies,  131 

Catalase,  92-3 

Centrifugation,  effect  on  sperm,  83 

Centriole,  14 

Centrosome,  13 

Cephalin,  124,  130 

Cervical  mucus,  penetration  test, 
40;  as  medium  for  sperm,  40,  54 

chloride,  effect  on  sperm,  57 

Cetyltrimethylammonium  bromide, 
(CTAB),  spermicidal  properties, 
50,  57;  effect  on  succinate  oxida- 
tion, 50 

Charcot-Leyden  crystals,  161 

Chemotaxis,  71-2 

Chiroptera,  copulatory  plug,  28 

Chloride,  18,21,23,32-3,  191 

Chloroform,  effect  on  sperm,  54 

Cholesterol,  86,  124-5,  130-2 

Choline,  119,  130,  168-74 

Choline  esterase,  173^ 

Chromatin,  in  sperm  nucleus,  11, 

Chromosin,  109 

Chromosomin,  109 

Citraconic  acid  and  chemotaxis,  72 

Citric  acid,  in  semen  and  reproduc- 
tive organs,  16-24,  31-3,  132, 
183-8,  191;  relation  to  andro- 
gens, 68,  139,  186;  metabolism 
and  function,  187-8;  association 
with  gel,  31,  34,  184,  188;  in 
diluents,  81;  in  prostatic  calculi, 

Citric  acid  test,  for  androgens,  68, 

Clam,  nucleoprotein,  100 

Clotting,  of  semen,  28-9,  114,  188 
Clotting  test,  for  androgens,  67 
Clupeine,  106-8 
Coagulating  gland  in  rat,  16,  29; 

fructose  secretion,    16,    138^5; 

transplantation,  144 
Coagulation  of  semen,  28-9,  114, 

Cock  semen  {see  also  fowl),  volume 

and  density,  2;  sperm  structure, 

11;  composition,  33;  pH,  44,  60; 

respiration,  50;  irradiation  effect, 

59;  diluents,  79-81;  nucleic  acid, 

105;  lipid  capsule,  126;  sugar,  137 
Cod-fish,  gadushistone,  108;  lipids, 

Coelenterate,  sperm  nucleic  acid, 

Cold  effect  on  sperm,  62-6 
Colostrum  corpuscles,  131-2 
Colour  {see  also  pigments),  1,  17, 

Conception   rate,  and  quality   of 

semen,  39,  42 
Contraception  {see  also  spermicidal 

substances),  55-8,  79-80,  97-8 
Contractive  substances,  in  sperm, 

14;  in  seminal  plasma,  27 
Copper,  88-9;  toxicity,  54,  58,  76 
Copulatory  plug,  28-9 
Corpora  amylacea,  131-3 
Cortical  helix,  15 
Cowper's  gland,  15,  35,  185 
Cozymase,  in  sperm,  157-8;  dephos- 

phorylation,  122 
Creatine,  179-81 
Creatinine,  179-81 
Crocetin,  dimethyl  esters,  in  Chlamy- 

domonas,  72 
Crocin,  in  Chlamydomonas,  72 
Crustaceans,  nucleic  acid,  104 
Cryptorchidism,  62 
Cumulus-dispersing     factor,     and 

hyaluronidase,  96 
Cyanide,  effect  on  sperm,  55,  56,  91, 




Cyclop  terin,  106 

Cyprinine,  106 

Cysteine,  effect  on  sperm,  58,  154, 

Cystine,  in  semen,  85,  87,  110, 

Cytochrome,  in  sperm,  14,  66,  87, 
90-2;  leakage  from  sperm,  10, 
57,  83;  role  in  metabolism,  90-2, 

Cytochrome  oxidase,  14,  87,  90-2 

Cytometer,  39 

Cytoplasm  of  sperm,  85-7;  baso- 
philic character,  5;  dehydration 
changes,  7 

Cytosine,  100-1 

Deer,  sperm  lipids,  6 

Dehydration  changes  in  sperm,  7-8 

7-Dehydrocholesterol,  133 

Dehydrogenase  activity,  48,  87,  92 

Dehydro/j'oandrosterone,  68-9 

Deoxyribonucleic  acid,  in  sperm, 
87-8,  98-107;  Feulgen  reaction, 
11,  100-1 

Deoxyribonucleoproteins  of  sperm, 
5,  8,  11,  86,  98-100,  106-9 

Deoxyribose,  100 

Deproteinizing  agents,  1 1 3 

Detergents,  spermicidal  properties, 
50,  57;  mode  of  action,  57 

Diabetes,  effect  on  seminal  fruc- 
tose, 146-8 

Diamine  oxidase,  167 

Diastase,  17 

Diazo  reaction,  175 

Dichlorophenol  indophenol,  reduc- 
tion, 23,  175,  179 

Diethyldithiocarbamate,  as  metal- 
binding  agent,  76 

Differential  (live-dead)  staining,  39, 

Diluents,  55,  73,  76,  79-82 

Dilution  effect,  on  sperm,  54,  73-9 

2  :  4-Dinitrophenol,  effect  on  sperm, 
52,  56 

1  :  6-Diphosphofructose,  dephos- 
phorylation,  36,  118-21,  150;  as 
intermediary  in  glycolysis,  38-9, 
150,  157 

Dirscherl-Zilliken  reaction,  68 

Disintegration  of  sperm,  86-7, 

Dodecylsulphate,  as  spermicidal 
agent,  50,  57 

Dog  ampullar  secretion,  adrenaline, 

Dog  prostate,  adrenahne,  181; 
choline-liberating  enzyme,  170; 
free  amino  acids,  20;  hyper- 
trophy, 20,  133;  corpora  amyl- 
acea,  131-3 

Dog  prostatic  secretion,  composi- 
tion and  properties,  17-18; 
enzymes,  17,  115;  lipids  and  lipid 
bodies,  126,  131-3;  effect  on 
sperm,  25 

Dog  semen,  volume  and  density,  2 
physico-chemical  properties,  28 
41-44;  composition,  33;  tempera 
ture  effect  on  sperm,  62,  63 
ethanol  effect  on  sperm  and  fer 
tihty,  56;  resistance  to  dilution 
78;  hyaluronidase,  94;  fibrino 
genase,  28;  acid  phosphatase,  29 
lipids,  126,  131;  fructose  and 
citric  acid,  33;  respiration,  50: 
effect  of  sugars  on  motility,  151-2 

Dogfish,  fructose,  136 

Dry  weight,  of  semen,  21,  32-3,  36, 

Echinarachnius  parma  (ssind-doWar), 
cyanide  effect  on  sperm,  92 

Echinochrome,  70 

Echinoderms,  (see  also  sea-urchin 
and  sea-cucumber),  sperm  nu- 
cleic acid,  104 

Echinus  esculentiis  {see  also  sea- 
urchin)  semen,  density,  74;  pH 
44;  dilution  effect,  74;  oxygen 
effect.  74;  glycogen,  5;  nucleic 


The  Biochemistry  of  Semen 

Echinus  esculentus  (contd). — 
acid,   104;  lipids  and  phospho- 
lipids,   125,    129;    sterols,    125; 
fatty  acids,   125;  adenosine  tri- 
phosphate, 155 

Echiurus,  creatine,  180 

Egg-membrane  lysin  in  molluscs,  95 

Egg-surface  liquefying  agent,  and 
androgamone,  23,  71 

Egg-water,  agglutinating  and  ac- 
tivating effects,  70-6,  114 

Egg-yolk,  diluents,  80;  eflfect  on 
sperm  respiration,  117;  glucose 
content,  157 

Ejaculate,  species  characteristics,  2; 
fractions,  9,  28,  35-8;  frequency, 

Ejaculatory  duct,  obstruction,  137- 

Electrical  impedance  changes,  41-2 

Electric  stimulation  of  ejaculation, 

Electro-conductivity,  41 

Electrolytes,  18,  23,  25,  32-3;  inter- 
relations, 42-4,  73-7,  188,  192 

Electron  microscopy,  application 
to  sperm,  11,  14-15 

Electrophoresis  of  seminal  pro- 
teins, 112 

End-piece,  characteristics,  15 

Enolase,  158 

Enzymes,  in  seminal  plasma,  15-29, 
114^23,  167,  170-3;  in  sperm,  5, 
13,  83-9,  117,  151-9,  173-4,  181- 
2;  release  from  damaged  sperm, 
10,  64,  83,  94-5;  in  metabolism, 
43-53,  128-30,  149-59,  187-8; 
aflfecting  sperm-fibrils,  14 

Epididymal  spermatozoa,  6-8,  24- 
5,  82,  86-8,  125,  129,  152-4, 

Epididymis,  as  storage  organ  for 
sperm,  6, 15,25, 35-7, 82, 1 54, 1 78, 
185;  secretion,  25,  35-6;  ripening 
effect,  25,  154;  dependence  on 
androgen,  67;  oxygen  tension,  61; 

citric  acid,  185;  dehydrocholes- 
terol,  133 

Epinephrine  (adrenalme),  181-2 

Ergothioneine,  in  semen  and  acces- 
sory secretions,  15,  20-3,  36-7, 
175-8,  191;  counteraction  of 
thiol-reagents,  58,  176-8;  role, 
176-8;  biogenesis,   178 

Esocine,  106 

Esox  lucius  (pike),  nucleic  acid,  105 

Ethanol,  effect  on  sperm,  56; 
excretion  in  semen,  56 

Ethylenediamine  tetra-acetate,  as 
metal-binding  agent,  76 

Ethylquinone,  effect  on  sperm,  57 

Evaluation  of  semen  quaUty,  38-53, 
78,  92-3,  138 

Exhaustion  effect,  34 

Extraneous  factors,  influence  on 
sperm,  54-82 

Fatty  acids,  in  semen,  125-7; 
utilization,  52 

Fatty  aldehydes,  in  acetal  phos- 
pholipids, 126 

Female  prostate,  187 

Female  reproductive  tract,  role  in 
sperm  transport,  8-9 

Fertility,  evaluation  through  semen 
analysis,  3,  38-53,  78-82,  92-5, 
138;  after  irradiation,  58-60; 
temperature  effects,  62-66;  of 
epididymal  sperm,  6-8;  and 
artificial  diluents,  79-82;  relation 
to  'lytic'  agents  and  hyaluroni- 
dase,  95-7;  in  malnutrition,  148- 
9,  172-3,  191;  contraception  and 
'anti-fertihty  factor',  55-8,  79- 
80,  97-8 

Fertilization,  8-10,  69-73,  95-8 

Fertilizin,  70-1 

Feulgen  reaction,  11,  100-1,  126-7 

Fibrils,  in  spermatozoa,  13-14 

Fibrin,  seminal,  114 

Fibrinogenase,  17,  29,  114-15 

Fibrinolysin,  17,  29,  114-15 



Fish  semen  {see  also  individual 
species),  pH  and  dilution  effects, 
60,  74-5;  proteins,  85-6,  106-9; 
amino  acids,  113;  lipids,  124-5; 
cholesterol,  124;  nucleic  acid, 

Fisswella  barbadensis  (limpet), 
sperm  nucleic  acid,  104 

Flagellum,  see  sperm-tail 

Flavin,  in  semen,  22,  33 

Flavonol  pigments,  in  plant  gam- 
etes, 72;  as  inhibitors  of  hyalur- 
onidase,  98 

Florence's  reaction,  168 

Fluoride,  effects  on  sperm  and 
enzymes,  50-2,  56,  121,  156,  158 

Foetal  fluids,  fructose,  134 

Formaldehyde,  effect  on  sperm,  57 

Forsythia,  cross-pollination,  72 

Fowl  semen  {see  also  cock  and 
turkey),  9,  11,  49,  59,  60,  65,  105 

Fox  semen,  2,  44,  50 

Fractions  in  semen  ejaculate,  35-8 

Freezing  of  sperm,  54,  63-5,  94 

Freezing  point,  and  osmotic  pres- 
sure of  semen,  34-5 

Frequency  of  ejaculation,  effect  on 
semen,  34-5 

Frog  sperm,  irradiation  effects,  59; 
pH  and  dilution  effects,  60,  73; 
survival  after  freezing,  63-4; 
cytochrome,  90;  nucleic  acid,  105 

Fructolysis,  46-53,  151-9;  index, 
46;  relation  to  sperm  density, 
motility  and  survival,  46-7,  151- 
3;  after  irradiation,  59;  after 
temperature-shock,  63;  inter- 
mediary enzymes  and  coenzymes, 
153-9;  effect  of  adrenaline,  182; 
effect  of  thiol  reagents  and  ergo- 
thioneine,  176-8 

Fructose,  in  semen  and  accessory 
organs,  16-24,  30-7,  135-8,  191; 
site  and  mechanism  of  formation, 
16,  121,  137-8,  149-151;  utiliza- 
tion, 37,  46-7,  52-3,   150-9;  in 

accessory  gland  transplants,  143- 
4;  in  gynogamone  mucoprotein, 
71;  in  diluents,  65,  78,  79;  rela- 
tion to  quality  of  semen,  138, 
143,  193;  relation  to  glucose, 
146-51;  relation  to  inositol,  192; 
relation  to  androgens,  139^9; 
after  castration,  and  hypophy- 
sectomy,  139-46;  after  scrotal 
application  of  heat,  62;  after 
vasectomy,  138;  in  diabetes, 
146-8;  relation  to  insulin,  146-8 
Fructosediphosphatase,  121 
Fructose  test,  for  androgens,  68, 

139^6,  186 
Fucaceae,  chemotropic  substances, 

Fucose,  in  gynogamone  mucopro- 
tein, 71 
Fumaric  acid,  and  chemotaxis,  72 
Fungi,  pigments  in  gametes,  72 

Gadushistone,  108 

Galactose,  in  gynogamone  muco- 
protein, 71 

Galea  capitis,  12-13 

Gamones,  69-73 

Gastrophilus  intestinalis,  fructose  in 
haemolymph,  136 

Gelatin,  in  semen  diluents,  80,  81; 
effect  on  sperm,  65 

Gelatin-liquefying  enzyme,  96 

Gelation  of  semen,  28-31,  35,  184 

Gillichthys  mirabilis  (goby),  protein 
in  the  vesicular  secretion,  22 

Glandula  vesicularis  in  rabbit,  21; 
fructose,  138;  citric  acid,  185 

Globulins,  seminal,  112 

Glucose,  in  semen  and  accessory 
secretions,  21,  33,  137;  utiliza- 
tion, 52-3, 150^-9;  in  gynogamone 
mucoprotein,  71;  in  diluents,  55- 
7,  65,  80-1;  relation  to  fructose, 
121,  146-51,  157;  in  egg  yolk 
and  follicular  fluid,  157;  relation 
to  inositol,  192 


The  Biochemistry  of  Semen 

Glucose  oxidase,  use  in  semen 
analysis,  136 

Glucuronidase,  in  the  prostatic 
secretion,  17 

Glutamic  acid,  in  the  prostate,  19, 
188;  in  mammalian  semen,  84, 
113;  in  fish  semen,  86;  in  semen 
diluents,  114;  role  in  transamina- 
tions and  citric  acid  formation, 

Glutamine,  in  the  prostate,  20 

Glutathione,  in  semen  and  acces- 
sory glands,  20,  33,  176-7; 
effect  on  sperm,  58,  154,  176-7 

Glycerol,  effect  on  sperm,  55;  use 
in  freezing,  64-6;  in  diluents,  81 

Glycerylphosphorylcholine,  16, 

Glycerylphosphorylcolamine,  126 

Glycine,  in  semen,  81,  85,  106, 
113-14;  protection  from  light 
and  dilution  effect,  114;  in 
diluents,  81,  114 

Glycogen,  5,  12,  129,  151-2 

Glycolysis,  46-8,  59,  151-4;  in 
semen  appraisal,  46-8;  after 
irradiation,  59;  in  epididymal 
and  ejaculated  sperm,  1 53-4 

Goat,  semen,  fructose,  136;  adrena- 
line, 181 

Goby,  proteins  of  the  vesicular 
secretion,  22 

Gomori's  method,  13,  118 

Gonadectomy,  effects,  113,  119, 
139-45,  186 

Gonadotrophins,  66,  68;  influence 
on  semen,  66,  68,  145-6,  148-9, 

Grasshopper,  fructose  in  reproduc- 
tive organs,  136 

Guanine,  100-1 

Guinea-pig,  accessory  secretions 
and  semen,  20,  23,  28,  126,  136, 
170,  172,  181-2;  fertility,  6,  62 

Gum,  in  diluents,  81 

Gynogamones,  70-1 

Haematin,  in  seminal  plasma,  22; 
in  sperm,  88-93 

Haemocuprein,  89 

Haemospermia,  22 

Haliotis  cracherodii  (abalone),  egg- 
membrane  lysin,  95 

Hamster,  fructose,  136;  inositol 
deficiency,  191 

Heat,  effect  on  sperm,  62 

Heavy  metal  compounds,  effect  on 
sperm,  57-8,  76,  98 

Hedgehog,  copulatory  plug,  28 

Heptacosane,   127-8 

Herring  semen,  nucleic  acid,  99 
nuclear  proteins,  99,  106-7: 
amino  acids,  113;  lipids,  124: 
agmatine  and  creatinine,  179 

Hesperidine,  98 

Hexokinase,  154-7 

Hexylresorcinol,  effect  on  sperm, 

Histidine,  84,  108 

Histochemistry  of  sperm,  5,  10-13, 
118,  126 

Histological  examination  of  semen, 

Histones,  98,  106-9 

Holothuria  tubulosa,  creatine  and 
creatinine,  180 

Hormones  {see  also  individual  hor- 
mones), effect  in  vivo  and  in  vitro, 
66-9,  139-45 

Human  semen,  see  man,  semen 

Hyaluronic  acid,  93,  96 

Hyaluronidase,  93-8;  release  from 
sperm,  10,  83,  94-5;  role,  96-8; 
inhibitors,  97-8;  relation  to 
citric  acid,  1 88 

Hydrocarbons,  chemotropic  acti- 
vity, 72;  in  semen,  127-8 

Hydrogen  ion  concentration,  in 
semen,  44—5;  changes  due  to 
glycolysis,  44;  relation  to  den- 
sity, motihty,  and  metabolism, 
44-5,  60-1;  in  accessory  secre- 
tions, 17,  22 



Hydrogen  peroxide,  toxicity,  58,  92, 
93;  formation  by  sperm,  58,  117, 
182;  and  X-ray  action,  58;  and 
oxygen  damage,  58 

Hydrogen  sulphide,  effect  on  sperm, 

Hydroxylamine,  effect  on  sperm,  91 

8-Hydroxyquinoline,  as  metal-bind- 
ing agent,  76 

Hyperpyrexia,  relation  to  azoo- 
spermia, 62 

Hypertonicity,  as  sperm-affecting 
factor,  61-2 

Hypophysectomy,  effect  on  semen, 
145-6,  186 

Hypophysis,  role  in  semen  forma- 
tion, 66,  145-9,  186 

Hypotonicity,  as  sperm-affecting 
factor,  61 

Immunology  of  semen,  79-80 

Impedance  changes,  frequency  in 
semen,  41;  relation  to  'wave 
motion'  and  conception  rate, 

India-ink  staining  method,  appli- 
cation to  sperm,  13 

Individual  variations  in  the 
composition  of  semen,  32-5,  137, 
184,  191 

Indophenol  reaction,  90-2 

Inhibitors  of  sperm  motility  and 
metabolism  {see  also  spermicidal 
substances),  55-8,  91-2,  97-8 

Inner  cylinder  in  the  axial  filament, 

Inosinic  acid,  dephosphorylation, 

Inositol,  in  semen  and  accessory 
secretions,  16,  21,  189-92;  role, 

Insectivora,  copulatory  plug,  28; 
corpora  amylacea,  132 

Insect  sperm,  transport,  and  sur- 
vival in  the  female  tract,  9;  PAS 
reaction,  12;  activation  by  semi- 

nal plasma,  25;  irradiation  effects, 

59;  fructose,  136 
Insulin,  effect  on  seminal  fructose, 

lodoacetamide    and    iodoacetate, 

effect  on  sperm,  56,  58,  158 
lodosobenzoate,  effect  on  sperm 

56,  58,  177-8 
lodospermine,  168 
Ions  and  ionic  equilibrium,  18,  23, 

25,  32-3,  42-4,  73-7,  188,  192 
Iron,  protein-bound  in  sperm,  88- 

93;    relation    to    haematin    and 

cytochrome,  89;  in  karyogen,  109 
Irradiation  effects,  58-60 
Isoagglutinin,  71 
Isoleucine,  in  mammalian  semen, 

84,  113;  in  fish  semen,  86,  106 

Jaffe's  reaction,  1 79-80 

Jelly-coat  of  eggs,  role  in  fertiliza- 
tion, 71;  dissolving-factor,  71, 

Jelly  fish,  sperm  nucleic  acid,  104 

Karyogen,  in  salmon  sperm,  109 

Keratin-like  protein,  in  sperm- 
membrane,  110 

Key-hole  limpet  (Megathura  crenu- 
lata),  egg-membrane  lysin,  95; 
nucleoprotein,  100 

Kinoplasmic  droplet,  6-7,  154 

Lactic  acid,  content  in  semen  and 
accessory  secretions,  21,  32-3, 
36;  as  product  of  fructolysis,  46, 
152-8;  as  substrate  for  sperm, 
52-3,  153 

Lactic  dehydrogenase,  158 

Lactose,  and  sperm  metabohsm,  55, 

Lead  salts,  toxicity,  54 

Leakage  of  proteins  from  sperm, 
10,  57,  83,  94^5 

Lecithin,  in  semen  and  accessory 
secretions,  84,  124-5,  130-2 


The  Biochemistry  of  Semen 

Leucine,  in  semen,  84,  113;  in 
herring  testes,  113;  in  diluents, 

Light  effects,  66-7,  91,  114 

Light-reflection  power  of  sperm,  8, 

Limpet,  nucleic  acid,  104 

Lipid,  124-33;  redistribution  dur- 
ing spermatogenesis,  6;  in  semen, 
32-3,  129-33;  in  sperm,  6,  86, 
124-7;  in  seminal  plasma  and 
accessory  secretions,  130-3;  meta- 
bolism, 49,  128-30,  172 

Lipid  'bodies',  in  semen,  131-2 

Lipid  capsule,  6,  126;  and  deter- 
gents, 57;  and  senescence,  77,  83 

Lipoprotein,  in  sperm,  15,  125-6; 
loss  due  to  damage  and  senes- 
cence, 77,  83,  84 

Lipositol,  in  L.  G.  B.-pabulum,  81; 
relation  to  inositol,  192 

Liquefaction,  of  semen,  28-9,  114; 
relation  to  citric  acid,  188 

Lithobius  forficatus,  cytoplasmic 
lipids  and  steroids,  6 

Live-dead  (differential)  staining, 
39,  60,  63 

Locusta  migratoria  (grasshopper), 
fructose  in  reproductive  organs, 

Longevity  of  sperm  {see  also 
survival),  9 

Lotahistone,  108 

Luminosity  of  sperm,  8,  41 

Lump-sucicer,  cyclopterin,  106 

Lysine,  in  seminal  proteins,  84,  108; 
free  in  human  semen,  113;  in 
herring  testes,  113;  in  diluents,  114 

Lytechinus,  dilution  effect  and 
amino  acids,  76;  sperm  nucleic 
acid,  104 

Lytic  agents,  92-8;  relation  to 
fertilization,  95-6 

Mackerel,  scombrine,  106;  scom- 
bron,  109 

Magnesium,  in  semen,  32-3;  in 
prostatic  calculi,  132;  relation  to 
motility,  and  metabolism,  77; 
effect  on  phosphatases,  121,  123 

Male  accessory  organs,  see  acces- 
sory organs 

Maleic  acid,  and  chemotaxis  in 
plants,  72 

Male  sex  hormone,  see  androgens, 
and  testosterone 

Malic  acid,  role  in  chemotaxis,  71 

Malnutrition,  effect  on  accessory 
organs  and  semen,  148-9 

Malonic  acid,  effect  on  sperm,  52 

Man,  prostate,  16,  19,  181-88 

Man,  prostatic  secretion,  physico- 
chemical  properties,  17-18;  con- 
tribution to  semen,  35;  enzymes, 
17,  114-15,  118-19,  170-1;  elec- 
trolytes, 17-18;  protein,  19;  lipids 
and  lipid  bodies,  130-2;  choles- 
terol, 130;  spermine,  166-7;  citric 
acid,  18,  183-8;  zinc,  19 

Man,  semen,  volume  and  density, 
2;  sperm  structure,  11,  14-15; 
physico-chemical  properties,  8, 
41-5,  60;  composition,  32-3; 
fractions,  35;  mineral  constitu- 
tents,  18-19,  32-3,  89,  188;  zinc 
and  carbonic  anhydrase,  19,  89; 
reducing  substances  and  ascorbic 
acid,  33,  48,  179;  fructose  and 
fructolysis,  32-4,  48,  136,  147- 
48;  coagulation  and  liquefaction, 
28,  188;  proteins  and  proteose  in 
seminal  plasma,  111-12;  mucoid 
substance,  112;  amino  acids, 
111-13;  spermine,  and  spermi- 
dine, 160-8;  choline  and  phos- 
phorylcholine,  130, 168-73;  adren- 
aline, 181;  sulphur  and  gluta- 
thione, 33;  lipids  and  lipid  bodies, 
101,  126,  130-2;  cholesterol,  130; 
oestrogen  and  androgen  content, 
68-9;  heptacosane  and  fatty 
acids,     127-8;   citric    acid,    17, 



183-8;  inositol,  191;  nucleic 
acid,  102;  Feulgen  reaction,  101; 
PAS  reaction,  112;  nuclease,  123; 
phosphatases,  117-23;  proteoly- 
tic enzymes,  114-16;  cytochrome, 
66,  90-1;  hyaluronidase,  94; 
amine  oxidases,  167, 182;  respira- 
tion, 41,  50,  90,  167;  choline 
esterase,  173;  hormonal  aspects, 
143-8;  freezing  effect,  63-5; 
effect  of  arsenicals,  58 
Man,  seminal  vesicle,  16,  35;  com- 
position of  secretion,  20,  23^, 
137;  monamine  oxidase,  182; 
phosphorylcholine,  171;  fructose, 
23^,  137 
Mannose,  as  substrate  for  sperm, 

52,  152,  154 
Manteau  lipidique,  15,  126 
Marsupialia,      copulatory      plug, 

Maturation  of  sperm,  see  ripening 
Megathura  cremdata  (key-hole  lim- 
pet), egg-membrane  lysin,  95 
Mercury  salts,  toxicity,  54,  57 
Mesaconic  acid,  and  chemotaxis,  72 
Metabolic  regulator,  153-4 
Metabolism,   chief  characteristics, 
45;  of  carbohydrate,  45-53,  151- 
9;  of  lipid,  128-30;  of  protein, 
114-17;  of  nitrogenous  bases,  1 67, 
169-73,  176-8,  180-2;   of  citric 
acid,  187-8;  extraneous  factors, 
55-9,  73,  79;  role  of  adenosine 
triphosphate,  129,  155-9 
Metal-chelating  agents,  and  sperm, 

76,  114 
Metalloproteins  in  semen,  88-93 
Methionine,  in  semen,  84,  87;  as 
substrate  for  ergothioneine  for- 
mation, 178 
Methoxyquinone,  effect  on  sperm, 

5-Methylcytosine,  100-1 
Methylene-blue  reduction  test,  48, 

Methylhydroquinone,     effect     on 

sperm,  57 
Meyerhof  oxidation  quotient,  153 
Middle-piece,  structure  and  com- 
position, 10,  13-14,  87;  separa- 
tion from  sperm-head  and  tail, 
86-87;  indophenol  reaction,  90-2; 
lipids,  126-30 
Milk,  as  diluent,  79,  81 
Milovanov's  diluent,  80 
Milovanov's  resistance  test,  78 
Mitochondrial  sheath,  6,  14,  126 
Mole,  copulatory  plug,  28 
Molluscs,  lytic  agents,  95;  sperm 

nucleic  acid,  104 
Monkey,    prostatic    phosphatase, 

Monoamine  oxidase,  182 
Motility    of   sperm,    types,    41-2; 
role  of  fibrils,  14;  assessment  and 
relation  to  fertility,  39-53;  rela- 
tion to  metabolism,  45-50,  91- 
2,  151-4;  activators  and  inhibi- 
tors, 25,  55-8,  92;  after  irradia- 
tion, 59;  dilution  effect,  72-82; 
role  of,  ions,  74,  77,  sulphydryl 
groups,  77,  176-8,  gamones,  69- 
72,  adenosine  triphosphate,  154- 
9,  spermine,  167,  acetylcholine, 
173,  ergothioneine,  176-8,  citric 
acid,  188 
Motility  test,  for  androgen,  67 
Mouse  sperm,  structure,  6,  7;  irra- 
diation effects,  59;  fructose,  136 
Mucopolysaccharase,  96 
Mucopolysaccharides,  12,  97 
Mucoprotein,  in  semen,  28,  33;  in 

sea-urchin  'jelly  coat',  71 
Mucus  plug,  in  the  honey-bee,  28 

Neck-piece  (neck,  sperm-neck),  13 
Necrospermy,  effect  on  fructolysis, 

Niacin  (nicotinic  acid),  33 
Nicotinamide  nucleotide,  dephos- 

phorylation,  122 


The  Biochemistry  of  Semen 

Niederland  reaction,  18 

Nitrogen,  total  and  non-protein, 
21,32-3,84-7,  111-13 

Nitrogen  mustard,  effect  on  sperm, 

Nitrogenous  bases,  in  semen, 

Noradrenaline,  in  semen,  181-2 

Nucleases,  123 

Nucleic  acid  {see  also  deoxyri- 
bonucleic and  ribonucleic  acid), 
in  developing  and  mature  sperm 
cells,  5;  detection  and  analysis,  5, 
86-8,  99-106;  species  charac- 
teristics, 101-6 

Nucleohistone,  see  nucleoproteins 

Nucleoprotamine,  see  nucleopro- 

Nucleoproteins,  5,  8,  11,  86,  98- 
100,  106-9 

5-Nucleotidase,  38,  121-2 

Nucleus,  as  part  of  sperm-head, 
7-12;  removal  from  sperm,  85; 
constituents,  98-109 

Nutrition,  effect  on  the  com- 
position of  semen,  148-9;  nutrient 
requirements  of  spermatozoa, 
xiii-xiv,  46-53,  128-9,  151-9 

Odour  of  semen,  relation  to  sper- 
mine and  spermidine,  165-6 

Oestrogens,  content  in  semen,  68; 
effect  on  phosphatases,  119; 
effect  on  seminal  fructose,  144-5 

Opossum,  clotting  of  semen,  28; 
fructose,  136 

Optical  properties  of  semen,  40-1 

Osmotic  properties  of  semen  and 
accessory  secretions,  7,  17-18, 
23,  43;  freezing  point  depression, 
43;  role  of  citric  acid,  188;  role  of 
inositol,  192 

Outer  cylinder  in  the  axial  filament, 

Ovarian  androgens,  144-5,  186 

Ovulase,  95 

Ovulation,  9 

Oxaloacetic  acid,  conversion  to 
citric  acid,  188;  as  substrate,  52 

Oxygen  damage,  58 

Oxygen  tension,  in  vas  deferens  and 
epididymis,  61;  effect  on  sperm 
motility,  73,  75,  151-2;  effect  on 
metabolism,  153 

Oxytocic,  hormone,  67;  properties 
of  seminal  plasma,  27 

Oyster  sperm,  glycogen,  6;  respira- 
tion, 50,  52 

Pantothenic  acid  in  semen,  33 

Paracentrotus  {see  also  sea-urchin), 
sperm  nucleic  acid,  104;  gelatin- 
liquefying  enzyme,  96 

PAS  reaction,  12,  112-13 

Pasteur  effect,  153-4 

Penicillin,  in  diluents,  81 

Pepsinogen,  116 

Peptides,  life-prolonging  effect  on 
sperm,  76;  as  products  of  pro- 
teolysis, 108,  111 

Perch,  106 

Percine,  106,  108 

Periodic  acid-Schiff  reaction,  12, 

Permeability  of  sperm,  10;  changes 
due  to  senescence  and  damage, 

pH,  see  hydrogen  ion  concentration 

Pharmacological  effects,  of  seminal 
plasma  and  accessory  gland 
secretions,  26-8;  of  alkaloids,  56; 
of  spermine,  162;  of  choline,  173; 
of  adrenaline  and  noradrenaline, 

Phenolphthalein  phosphate,  as  sub- 
strate for  phosphatase,  1 1 8 

2-Phenoxyethanol,  spermicidal  ac- 
tion, 50 

Phenylalanine,  in  semen,  84,  113; 
oxidation,  117 

p-Phenylenediamine,  oxidation, 



Phenylmercuric  acetate,  effect  on 
sperm,  57 

Phenylphosphate,  as  substrate  for 
phosphatase,  118 

Phosphatases,  1 17-23;  see  also  acid 
and  alkaline  phosphatase,  5- 
nucleotidase,  adenosinetriphos- 
phatases  and  pyrophosphatase 

Phosphate  {see  also  phosphorus 
compounds),  in  semen  and  ac- 
cessory secretions,  18,  21,  32-3, 
107;  in  prostatic  calculi,  132;  in 
diluents,  77-8,  80-1;  effect  on 
sperm,  77;  formation  by  phos- 
phatases, 117-23,  167,  171-2; 
esterification,  158 

Phosphoarginine,  in  sperm,  181 

Phosphocreatine,  in  sperm  and 
testes,  180-1 

Phosphofructokinase,  157 

1-Phosphofructose,  120,  121, 

6-Phosphofructose,  118,  120,  121, 
150,  157 

Phosphoglucomutase,  150-1 

1-Phosphoglucose,  120,  150-1 

6-Phosphoglucose,  118,  120,  150-1, 

Phosphoglyceric  acid,  158 

Phosphoglycerol,  117 

Phosphohexose  isomerase,  121, 
150-1,  157 

Phospholipids,  in  mammalian  se- 
men and  accessory  secretions, 
32-3,  107,  125-3;  in  fish  sperm, 
124-5;  metabolism,  127-30,  172 

6-Phosphomannose,  120 

Phosphopyruvic  acid,  1 58 

Phosphorus  compounds  in  semen 
and  accessory  secretions,  20-1, 
32-3,  36,  84-8,  99,  103,  107, 
129-30,  155-9,  167,  170-3;  see 
also  under  individual  compounds 

Phosphorylations,  in  lipid  meta- 
bolism, 129;  in  carbohydrate 
metabolism,  10,  149-59 

Phosphorylcholine,  in  semen  and 
accessory  secretions,  16,  119,  168, 

Phosphorylethanolamine,  in  pros- 
tate, 20 

Phosphorylhesperidine,  98 

Phosphotriose,  oxidation  by  sperm, 

Phosphotriose  dehydrogenase,  158 

Physico-chemical  properties  of  se- 
men, 40-5 

Picrocrocin,  in  Chlamydomonas,  72 

Pigments,  22,  70,  72,  90-2 

Pike,  nucleic  acid,  105;  esocine,  106 

Pilocarpine,  effect  on  prostate,  18 

Pituitary  gland,  role  in  semen  for- 
mation, 66,  145-9 

Plants,  chemotaxis  and  sperm-egg 
interacting  substances,  71-2; 
sperm  cytochrome,  92 

Plasmal,  101-2,  126-7 

Plasmalogens,  126-7 

Plasmin,  115 

Plasmolysis  of  spermatozoa,  by 
enzymes,  14;  by  chemical  agents, 
14,  85-6,  98 

Poisons,  effect  on  sperm,  55-8;  see 
also  inhibitors  and  spermicidal 

Polymixin,  in  diluents,  81 

Porgy,  sperm  lipids,  124 

Post-sperm  fraction,  35-8 

Potassium,  in  accessory  secretions, 
18,  21-3;  in  mammalian  semen, 
32-3,  36;  in  fish  and  sea-urchin 
semen,  43^;  relation  to  citric 
acid,  44,  188;  effect  on  sperm,  55, 

Preputial  glands,  7-dehydrocholes- 
terol,  133 

Pre-sperm  fraction,  33-8 

Progesterone,  androgenic  effect, 

Prohn,  in  semen,  85,  106,  113 

Propionic  acid,  as  substrate,  52 

Prostaglandin,  26-7 


The  Biochemistry  of  Semen 

Prostate  gland,  secretory  function, 
16-20,  130-3, 168-73,  181, 184-8, 
zinc,  19;  phosphatases,  117-19; 
corpora  amylacea,  131-3;  amino 
acids  and  transaminases,  19-20, 
188;  in  the  female,  187 

Prostatic  calculi,  131 

Prostatic  cancer,  119 

Prostatic  phosphatase,  see  acid 

Prostatic  secretion,  species  charac- 
teristics, 16-20,  130,  184-5;  pH, 
17;  effect  on  sperm,  25;  enzymes, 
17,  114-19;  mineral  constituents, 
18;  fructose,  16,  138;  citric  acid, 
16-19,  183-8;  lipids,  130-3; 
amino  acids,  19,  spermine,  166-7 

Protamines,  98-9,  106-9 

Proteins,  extracellular,  19,  22,  76, 
84,  111-23;  intracellular,  14,  43, 
83-110,  124;  release  from  sperm, 
10,  57,  74,  83;  in  coagulation  and 
liquefaction,  28-34, 114-15;  effect 
on  sperm,  55,  64,  76,  79 

Proteolytic  enzymes,  in  semen  and 
reproductive  organs,  17-19,  29, 
96,  114-16;  effect  on  sperm,  14 

Proteose,  seminal,  19,  22,  111-14 

Psammechinus  {see  also  sea-urchin), 

Pseudo-hypophysectomy,  effects, 
148-9,  186 

Pteridium  aquilinum,  motility  and 
cytochrome,  92 

Puranen  reaction,  165 

Pyrophosphatase,  122 

Pyrophosphorylcholine,  171 

Pyruvic  acid,  metabolism,  50,  51, 
158;  as  substrate,  50-2,  158 

Quality  of  semen,  relation  to  other 

properties  of  semen,  metabolism 

and  fertility,  38-53,  78,  92-3, 138 

Quercetin  glycosides  in  pollen,  72 

Quinones,   spermicidal  properties, 

57,  effect  on  hyaluronidase,  98 

Rabbit  ampullar  secretion,  138, 185 
Rabbit  glandula  seminalis,  21 
Rabbit  glandula  vesicularis,  16,  21, 

138,  172,  185 
Rabbit  prostate,  16;  fructose,  16, 
138,     144-5;     adrenaline,     181; 
citric  acid,  185 
Rabbit  semen,  volume  and  density, 
2,     31;     sperm     structure,     11; 
motility  and  fertility,  in  relation 
to  extraneous  factors,  6,  9,  56, 
59-62,  78,  97;  physico-chemical 
properties,  41-^4,  60-2;  gel,  28, 
34;  fructose,  32,  34, 136-7, 140-7; 
citric  acid,    184-6;   lipids,    126, 
131;  hyaluronidase,  94;  respira- 
tion, 49-50;  castration  and  hor- 
mone effects,  140-6 
Rabbit  testis,  choline,  170 
Radiation  effects,  58-60 
Radioactive  compounds,  178 
Ram  semen,  volume,  density  and 
composition,      2,      32-3,      107; 
sperm  structure  and  disintegra- 
tion, 11,  87,  110,  126;  time  re- 
quired to  reach  the  ovum,   9; 
nucleic  acid,  55,   88,   102,   107; 
proteins,  88-110,  113;  ammonia, 
32,  116;  glycogen,  5;  fructose  and 
fructolysis,    32-4,    136,    152-9; 
enzymes,  87,  93,   120-2,  150-9, 
174;  lipids,  107,  126;  respiration 
and  cytochrome,  49-53,  87,  90-1; 
phosphorus     compounds,     107, 
155;  citric  acid,  184;  temperature 
effect,  62;   dilution  effect,  77-9; 
irradiation  effect,  59 
Rat    coagulating    gland,    fructose 
formation,   16,   138-44;  role  in 
coagulation,  29 
Rat  preputial  gland,  dehydrocho- 

lesterol,  133 
Rat  prostate,  structure  and  func- 
tion, 16,  19,  138,  184-5;  in  the 
female,  187;  fructose,  19,  138-44; 
citric  acid,  19,  185-7;  zinc  and 



carbonic  anhydrase,  19,  20; 
amino  acids  and  amines,  20; 
phosphatase,  120;  transaminase, 
188;  castration  and  hormone 
effects,  120,  141-9,  186-7;  trans- 
plantation, 143-4 

Rat  semen,  sperm  structure,  11; 
lipids,  6;  copulatory  plug,  28; 
choline,  1 69-70;  irradiation  effect, 

Rat  seminal  vesicle,  16,  187;  com- 
position of  secretion,  20,  172, 
185-7;  hormone  effects  and  trans- 
plantation, 120,  186 

Reducing  substances,  20-4,  33,  48, 
135,  174-9;  see  also  ascorbic 
acid,  fructose,  ergothioneine 

Refractive  index  of  sperm,  8 

Reptiles,  94,  105 

Resistance  to  dilution,  78 

Respiration  of  sperm,  49-53,  91-2, 
117,  129-31,  152-4,  167;  sub- 
strates, 50-3  ;  inhibitors,  49-51, 
56;  after  irradiation,  59;  pH- 
optimum,  60;  after  temperature- 
shock,  63;  in  presence  of  'egg- 
water',  76 

Rhinolophidae,  copulatory  plug, 

Riboflavin,  22,  33 

Ribonucleic  acid,  5,  86 

Ring  centriole,  14 

Ringer  solution,  as  semen  diluent, 

Ripening  of  sperm,  4-8,  25,  41-3, 
90,  153-4,  178 

Rodents  {see  also  guinea-pig,  rab- 
bit, rat),  clotting  phenomenon  in 
semen,  28 

Rutin,  in  pollen,  72 

Salmine,  106,  108 

Salmofario  {see  also  trout),  nucleo- 

protein  and  nucleic  acid,  99,  105 
Salmo   fontinalis,    constituents    of 

seminal  plasma,  84 

Salmon  semen,  mineral  consti- 
tuents, 43,  84;  proteins,  84-6, 
98-9,  106-9,  125;  nucleic  acid, 
86, 103;  lipids  and  cholesterol,  86, 
124-5;  free  amino  acids,  86;  cata- 
lase,  92;  phosphatases,  86;  andro- 
gamones,  71 

Salt  effects  on  sperm,  55,  62 

Sand-dollar,  cyanide  effect  on 
sperm,  92 

Saponin,  effect  on  sperm,  57 

Saxostrea  commercialis  (oyster), 
respiration  of  sperm,  50 

Scherer-Salkowski  reaction,  190 

Schiff's  reagent,  11,  126 

Scombrine,  106 

Scombrone,  109 

Scylliorhinus  caniculus  (dogfish), 
fructose,  136 

Sea-cucumber,  sperm  nucleic  acid, 

Sea-urchin  semen  (see  also  Arbacia, 
Echinus,  Lytechinus,  Paracentro- 
tus,  Psammechinus,  Strongylocen- 
trotus),  density,  74;  pH,  44;  potas- 
sium, 44;  glycogen,  6,  129;  nucleic 
acid,  100,  104;  nuclease,  123; 
proteins,  84,  108;  proteolytic 
enzymes  and  'lytic  agents',  95-6; 
sperm-egg  interacting  substances, 
69-71,  95-6;  lipids  and  sterols, 
125, 129;  adenosine  triphosphate, 
155;  creatine  and  creatinine,  180; 
cytochrome,  90-2;  catalase,  93; 
copper,  90;  effects  of  radiation, 
oxygen,  dilution  and  other  ex- 
traneous factors,  52-9,  74-7, 
91-2,  114;  metabolism,  50,  52, 
56,  74,  76,  91-2,  129-31,  151 

Semen  composition,  tables,  2,  20, 
21,  32-6,  84-90,  103-7,  130,  169, 
185,  191 

Seminal  duct,  7,  15,  61,  138 

Seminal  plasma,  origin,  composi- 
tion and  function,  15-29;  separa- 
tion from  sperm,   83;  proteins, 


The  Biochemistry  of  Semen 

Seminal  plasma  {contd.) — 
amino  acids  and  enzymes,  lll- 
23;  lipids,  130-3;  fructose,  135-59; 
nitrogenous  bases,  160-82;  citric 
acid,  183-8;  inositol,  189-92 

Seminal  stains,  detection,  163-8 

Seminal  vesicles,  secretory  func- 
tion, 15-24,  35-8,  191;  amino 
acids,  113;  phosphatases,  120-2; 
lipid,  131-3;  fructose,  137-8, 
142;  choline,  167-73;  ergothio- 
neine,  175-6;  citric  acid,  184-8; 
inositol,  190-2;  role  in  coagula- 
tion, 29 

Seminiferous  epithelium  {see  also 
spermatogenesis),  4,  59,  94 

Senescence,  12,  73-9,  83 

Serine,  85,  106,  113 

Sertoli  cells,  glycogen,  5;  lipids,  6 

Snail,  sperm  nucleic  acid,  104 

Sodium,  18,  21,  32-3,  36,  43,  188 

Specific  gravity,  7-8,  42-3 

Spermatheca,  9 

Spermatic  veil  ('floating  cap'),  12 

Spermatids,  origin,  1-4;  glycogen,  5 

Spermatin  (spermine),  161 

Spermatocytes,  basophilic  cyto- 
plasm, 1-4;  glycogen,  5;  lipid,  6 

Spermatogenesis,  accompanying 
chemical  phenomena,  4-8,  109; 
onset,  62,  140-1,  186;  length, 
178-9;  dependence  on  hormones, 
nutrition  and  other  factors,  62, 
66-7,  72,  143,  148-9;  after 
irradiation,  59 

Spermatogonia,  4-6 

Spermatophore,  9 

Spermatozoa,  1-15,  38-45;  separa- 
tion and  disintegration,  83-8; 
metabolism,  45-54,  117,  128-33, 
151-9,  177-8;  nucleic  acid  and 
proteins,  83-123;  lipids,  125-30 

Spermatozoids  in  mosses  and  ferns, 

Sperm-density,  species  characteris- 
tics,  1-2,  31-6,  82;  relation  to 

other  properties  and  metabolism 
of  semen,  39-53,  138,  193 

Sperm-egg  interacting  substances, 

Sperm-head,  10-13,  85-6,  94,  98- 
110,  126 

Spermicidal  substances,  50,  54-8, 
79-80,  97-8 

Spermidine,  166-7 

Spermine,  2,  160-8 

Sperm-lysin,  71,  95-6 

Sperm-maturation,  see  ripening 

Sperm-membrane,  13,  110 

Sperm  middle-piece,5eemiddle-piece 

Sperm-rich  fraction,  35-8 

Sperm-ripening,  see  ripening 

Sperm-tail,  10,  14-15,  85-7,  124-6 

Spermosin,  14 

Sphingomyelin,  124 

Spiral  body,  14,  126 

Split-ejaculate  method,  use  in 
semen  analysis,  35-8 

Staining  methods,  12-13,  39,  60,  63, 
100-1,  126 

Stallion  reproductive  organs,  cho- 
line, 168;  citric  acid,  184 

Stallion  semen,  volume  and  dens- 
ity, 2;  abnormalities,  11;  physico- 
chemical  properties,  41-4;  frac- 
tions, 38;  gelation,  28;  composi- 
tion, 32-3;  androgen  content,  69; 
citric  acid,  184;  fructose,  32,  136; 
choline,  170;  creatine  and  creati- 
nine, 180;  freezing  and  dilution 
effects,  63,  78 

Starfish  sperm,  92,  114 

Sterility,  see  fertility 

Steroids  in  semen  and  reproductive 
organs,  6,  67-9,  86,  124-5,  130-3; 
urinary  excretion,  62 

Stichopus  diabole  (sea-cucumber), 
sperm  nucleic  acid,  104 

Stilboestrol,  144-5 

Storage  of  sperm,  in  the  epididymis, 
6-7;  in  the  female  tract,  8-9;  with 
diluents,  79-82 



Streptomycin,  in  diluents,  81 

Strongylocentrotiis,  (see  also  sea- 
urchin),  114,  180 

Sturgeon  sperm,  104,  106,  108 

Sturine,  106,  108 

Succinic  acid,  oxidation,  50,  52,  57, 
87,  92 

Succinic  dehydrogenase,  87,  92 

Sucrose,  effect  on  sperm,  55,  152; 
use  in  freezing,  64,  65;  in 
chemotaxis,  71 

Sulphate,  21,  77,  175-8 

Sulphite,  179 

Sulphonamides,  excretion  in  semen, 
57;  in  diluents,  81 

Sulphur,  elementary,  154;  com- 
pounds, 21-3,  33,  56-8,  71,  77, 
84-7,  110,  154,  176-9 

Sulphydryl-binding  substances,  56- 
8,  176-8 

Sulphydryl  groups,  function,  23, 
56-8,  77,  154,  176-9 

Surface-active  substances,  spermici- 
dal properties,  50,  57 

Survival  of  sperm,  6-9,  24-5,  60-1, 
66-7,  78-82,  151-4 

Tail-sheath  (see  also  sperm-tail), 

Taurine,  in  prostate,  20 

Temperature,  effects  on  sperm,  12, 

Temperature  shock,  12;  preven- 
tion, 80 

Tench,  nucleic  acid,  105 

Testis,  role  in  sperm  formation, 
4-6;  spermine,  162;  choline,  170; 
creatinine,  179;  cytochrome,  90; 
hyaluronidase,  94;  phosphocrea- 
tine,  180-1 

Testosterone,  69;  effect  on  the 
composition  of  semen,  113,  139- 
45,  185-7 

Thiamine,  33 

Thioglycolic  acid,  effect  on  sperm- 
membrane,  110 

Thiol-groups,  function,  23,  56-8, 
77,  154,  176-9 

Thiolhistidine,  relation  to  ergo- 
thioneine,  174,  178 

Thiol-reagents,  56-8,  176-8 

Threonine,  84,  113 

Thymine,  100,  101 

Thynnine,  106 

Thyroxine,  effect  on  sperm,  68,  71 

Toluquinone,  effect  on  sperm,  57 

Tonicity,  as  sperm-affecting  factor, 

Transaminase,  19,  186 

Transferase  activity  of  phospha- 
tase, 119 

Transmethylations,  172,  178 

Transplants  of  accessory  organs, 
secretory   function,    143-4,    186 

Transport  of  spermatozoa,  in  the 
male  and  female,  6-9,  178-9 

ethanol,  effect  on  sperm,  57 

Trimethylbenzylammonium  chlor- 
ide, effect  on  sperm-membrane, 

Triphenyltetrazolium  chloride, 
effect  on  sperm,  56 

Trithiolpropane,  effect  on  sperm, 

Trout  semen,  nucleic  acid  and 
nuclear  proteins,  99-109;  potas- 
sium, 43,  74;  effects  of  oxygen, 
dilution  and  ions,  73-4 

Tryptic  enzyme,  1 1 5 

Tryptophan,  in  semen,  84,  109; 
oxidation,  117 

Tunny  fish,  thynnine,  106 

Turbidity  of  semen,  40-1 

Turkey  semen,  volume  and  density, 

Turpentine,  effect  on  sperm,  54 

Tyrosine,  in  semen,  85,  86, 108, 113; 
oxidation,  117 

Ultrasonic  waves,  use  for  sperm 
disintegration,  86,  100 


The  Biochemistry  of  Semen 

Urea,  in  semen,  32,  116;  effect  on 
sperm,  55 

Urechis  caupo,  creatine  and  creati- 
nine, 180 

Urethral  glands,  15,  37-8 

Uric  acid  in  semen,  32,  123 

Vitamins,  role  in  reproduction 
and  semen  formation,  23,  72, 

Vitrification,  64-6 

Volume  of  semen,  1-2,  31-6 

Washing,  effect  on  sperm,  79-84 
Wave  motion  in  semen,  41 
Witte's  peptone,  in  semen  diluent, 

Wolffian  duct,  21 

Xanthine  oxidase,  22,  123 
X-rays,  effect  on  sperm,  58-9 

Zinc,  in  semen  and  prostate,  19, 

Zymohexase,  157